BACKGROUND
interest in the cellular and molecular mechanisms of regeneration has stimulated a resurgence of investigations in a growing cohort of organisms  <cit> .
for example, planarians  can recover from nearly any plane of transection, re-establishing their body plan and rebuilding internal organs such as the nervous and digestive systems within a week after injury  <cit> .
recent investigations have illuminated some of the mechanisms of planarian regeneration, including the re-establishment of axial polarity  <cit> , somatic stem cell dynamics  <cit> , tissue remodeling  <cit> , organogenesis  <cit> , reproductive maturation and germ cell development  <cit> , and the molecular nature of regenerative competence  <cit> .
as in many experimental animal models  <cit> , these advances have required the adaptation and development of a range of tools and techniques, including methods for visualizing specific organs, tissues, and cell types.
in particular, optimization of protocols for in situ hybridization  <cit>  and sample processing for electron microscopy  <cit>  have dramatically increased the resolution of regenerative events at the cellular level.
by contrast, a rigorous analysis of the influence of specific steps during sample preparation for immunofluorescence has not been undertaken, and the collection of planarian-specific antibodies remains limited.
development of more systematic approaches for testing the effects of specific parameters on immunolabeling by individual antibodies would accelerate the generation of cell- and tissue-specific reagents, and expedite studies of post-transcriptional aspects of gene expression during regeneration, such as protein localization and modification.
historically, characterization of planarian tissues and studies of their responses to injury were conducted using histological stains, vital dyes, and electron microscopy  <cit> .
more recently, significant improvements to in situ hybridization  protocols  <cit>  have enabled the use of rna probes to label organs, subpopulations of cells, and ribonucleoprotein particles  <cit> .
in addition to these methods, protocols utilizing both lectins and antibodies as cell-specific probes have also been developed.
these protocols are less labor intensive and more economical than ish protocols, and, in addition to detection of specific cell types, enable resolution of subcellular regions such as membranes, nuclei, and neuronal processes.
a variety of lectins and antibodies  have been generated or identified that label the secretory system  <cit> , reproductive system  <cit> , nervous system  <cit> , intestine  <cit> , protonephridia  <cit> , muscles  <cit> , and stem cells  <cit> .
in addition, commercial and publicly available antibodies that cross-react with planarian antigens in the nervous system and other tissues have been identified  <cit> .
although antibodies are most often raised against specific molecules  <cit> , monoclonal antibodies  have also been generated in large-scale screens using purified cells, tissues, or whole-animal homogenates as immunogens.
such screens have yielded specific markers for neurons and their projections  <cit> , regenerating tissues  <cit> , and other cell types  <cit>  in a variety of organisms.
tissue-based mab screens bypass potential difficulties such as the need to identify highly expressed proteins or immunogenic regions appropriate for production of fusion proteins.
additionally, mab screens result in the generation of clonal, immortal hybridoma lines and therefore a theoretically inexhaustible supply of antibody  <cit> .
several mab screens have been conducted using planarian cells and extracts, and have yielded markers for various tissues and cell types  <cit> .
despite recent progress, the repertoire of antibodies that recognize planarian tissues is still limited, and a greater collection of reagents that label cell types unique to specific organ systems is needed.
for example, regeneration of the intestine requires both remodeling of pre-existing, post-mitotic intestinal tissue and addition of new intestinal cells at the growing ends of regenerating gut branches  <cit> .
antibodies currently used to label the intestine  <cit>  lack specificity, labeling additional tissues such as pharynx, epidermis, and nervous system.
furthermore, some of these antibodies label only subregions of intestinal cells such as the apical surface, making them less than ideal for analysis of remodeling and growth during intestinal morphogenesis.
in order to develop more specific intestinal antibodies, we took advantage of a protocol we recently developed  <cit>  that enables purification of intestinal cells, and conducted a mab screen using these purified cells as immunogens.
we generated ten mabs that labeled the intestine, as well as  <dig> mabs that label the nervous system, epidermis, secretory cells, and other cell types.
because sample processing is known to influence antibody-antigen interactions  <cit> , we also systematically evaluated the effects of various parameters during fixation of planarian tissue, including chemical treatments to remove mucus and pigmentation.
this analysis led to the identification of optimal sample preparation protocols for several mabs, and to the development of an optimization workflow that efficiently tests the influence of multiple processing steps on immunolabeling in planarians.
RESULTS
a monoclonal antibody screen utilizing purified intestinal phagocytes as immunogens
the planarian intestinal epithelium is comprised of two cell types: secretory goblet cells that release digestive enzymes after food ingestion, and absorptive phagocytes that engulf food for intracellular digestion  <cit> .
intestinal phagocytes retain dyes, beads, and other compounds for up to several weeks after they are ingested  <cit> .
we recently developed a protocol in which animals are fed iron beads and dissociated into single cell suspensions, enabling the purification of phagocytes by magnetic sorting   <cit> .
using this protocol , we collected phagocytes from more than  <dig> planarians.
after fixation, these cells were used as immunogens for a monoclonal antibody screen.figure  <dig>
generation of monoclonal antibodies that recognize the planarian intestine and other tissues.
screen strategy.
preimmunization serum  did not detectably label planarian tissue.
test sera  ubiquitously labeled planarian tissues, with slightly elevated intestinal labeling.
pie chart depicting the number of clones  in the primary screen with indicated labeling specificities.
examples and schematics of immunolabeling from the primary screen.
mabs that label the intestine .
mabs that label the nervous system .
examples of mabs that label other non-intestinal tissues .
anterior is to the top in all images.
cell and tissue types are indicated in each panel.
“cns”, central nervous system.
“vnc”, ventral nerve cord.
scale bars:  <dig> μm ;  <dig> μm ;  <dig> μm .
three mice were immunized; test bleeds from all three mice labeled fixed planarians, while preimmune sera did not .
immunofluorescence was ubiquitous, likely reflecting the fact that many intestinal antigens are also expressed in other planarian cell types .
we also observed slightly elevated signal in the intestine for all three test sera .
one mouse was chosen for final immunization  and hybridoma generation.
after fusion of splenocytes with myeloma cells and cloning of the resulting hybridomas, supernatants from  <dig> primary lines were screened for immunoreactivity with fixed planarians .
in total,  <dig>  supernatants labeled planarian tissues at levels visibly higher than secondary antibody controls .
seventy  supernatants labeled the intestine .
in addition,  <dig> supernatants  labeled non-intestinal tissues .
these included pigment cells of the optic cup , neurons and their processes , muscles , epidermis , secretory cells  , and cilia .
twenty-four  antibodies that labeled non-intestinal tissues also labeled the intestine , suggesting the recognition of highly immunogenic antigens that are expressed at lower levels in the intestine than in other tissues.
alternatively, some non-intestinal antibodies might also have been generated against antigens expressed by contaminating cells or cell fragments in intestinal cell preparations.
in previous monoclonal screens, less than 8% of antibody-producing clones labeled intestinal tissue  <cit> .
by contrast, in our screen ~25% of positive clones  labeled the intestine with marked specificity.
because intestinal cells comprise only 3-8% of all planarian cell types  <cit> , these results show that immunization with an enriched cell population is an efficient strategy for generating cell type-specific mabs.
we retained  <dig> hybridomas, giving preference to those that produced antibodies labeling the intestine and other specific cell types with high signal and low background .
<dig> of  <dig> hybridoma lines produced igm class antibodies, while two lines produced igg mabs .
igm-producing hybridomas can dominate an immune response due to underimmunization with suboptimal amounts of antigen, or an abundance of complex carbohydrates and lipids when whole cells are used as an immunogen  <cit> .
consistently, a recent effort to generate mabs against membranes purified from planarian stem cells yielded many igm-producing lines  <cit> .
although igm mabs sometimes have lower affinity, reduced specificity, and decreased ability to penetrate tissue  <cit> , our screening strategy identified igm mabs that labeled specific cell types and penetrated tissues well.
each of the  <dig> hybridomas was subcloned, and we identified at least one unique line that continued to secrete high levels of specific mab after rescreening, freezing, and expansion.
subclones were deposited in the developmental studies hybridoma bank  for archival and public distribution.table  <dig>
monoclonal antibodies, isotypes, and tissue specificity
clone
isotype*
tissue recognized
*all mabs possessed kappa light chains.
†pattern based on primary screen; mab has not been characterized further.
optimization of planarian relaxation, mucus removal, and fixation improves immunolabeling
although our mabs consistently labeled specific tissues in the primary screen, levels of both signal and non-specific background often varied from animal to animal and within different regions of individual animals.
because epitope accessibility and antibody specificity depend critically on fixative choice and other sample processing steps  <cit> , we hypothesized that optimization of fixation and other treatments would improve labeling with some mabs.
processing of planarian tissue for immunofluorescence requires several steps in addition to fixation, including chemical removal of mucus secretions prior to fixation, bleaching to remove pigmentation and enable visualization of internal structures, and post-fixation treatments to increase tissue permeability and expose antigens .
a range of approaches to these steps have been developed to prepare planarians for whole mount in situ hybridization, immunolabeling, and histological analysis  <cit> .
in our primary screen, we tested hybridoma supernatants on hcl-treated, formaldehyde-fixed planarians that were bleached in hydrogen peroxide diluted in pbs.
because formal demonstrations of the effects of specific treatments at each stage of sample processing are rarely reported, we evaluated additional approaches to these steps simultaneously by testing combinations of treatments together .figure  <dig>
sample processing influences immunofluorescent labeling of the planarian intestine.
overview of steps during fixation and processing.
mab 4d <dig> labeling.
mab 3g <dig>   mab 2g <dig>  for all three mabs, labeling was best  in animals relaxed in magnesium and fixed with methacarn .
in some samples, magnesium treatment also improved labeling of anterior intestinal branches .
“mg”, magnesium-induced relaxation.
“nac”, n-acetyl-l-cysteine treatment.
“hcl”, hcl treatment.
“fa”, formaldehyde/triton x- <dig> fixation.
“mcn”, methacarn fixation.
“tsa”, tyramide signal amplification.
samples were bleached in methanol/6% h2o <dig> .
scale bars:  <dig> μm.
we began by comparing two chemical treatments commonly used to relax planarians and remove their mucus secretions prior to fixation: hydrochloric acid   <cit>  and n-acetyl-l-cysteine   <cit> .
we also compared formaldehyde, a cross-linking fixative  <cit> , with methacarn, a coagulating fixative  <cit> , reasoning that antibodies raised against cells fixed in formaldehyde and methanol would be most likely to react with planarian tissues that had been fixed similarly.
additionally, we tested magnesium chloride, which has been used to relax marine and freshwater invertebrates prior to fixation  <cit> , as an extra step prior to mucus removal.
we have previously utilized magnesium-induced relaxation to increase resolution of individual branches of the planarian intestine  <cit> .
for these optimizations, we utilized indirect detection , which dramatically improved signal intensity for supernatants generated after the primary screen .
furthermore, blocking time was increased to overnight , a step that moderately improved signal-to-noise for some intestinal antibodies , without affecting labeling efficiency for others .
for intestine-specific mabs, magnesium treatment universally improved labeling .
for example, in the absence of magnesium treatment, mabs 4d <dig> and 3g <dig> labeled anterior and posterior intestinal branches in hcl-treated, formaldehyde-fixed planarians, but penetration in the prepharyngeal region was poor .
when animals were relaxed in magnesium prior to hcl treatment, labeling of the primary anterior intestinal branch was dramatically improved.
similar results were observed for a third mab, 2g <dig>  in methacarn-fixed planarians .
mucus removal method also affected intestinal labeling, albeit more moderately.
for example, hcl treatment improved signal and reduced non-specific labeling by antibodies 4d <dig> and 3g <dig>  in formaldehyde-fixed planarians, while nac treatment led to slightly more specific labeling by 2g <dig> .
we also attempted to fix nac-treated animals with methacarn, but animals disintegrated during bleaching .
milder nac treatments or reduced h2o <dig> concentration during bleaching improved integrity, but abolished intestinal labeling .
although we cannot rule out that some antibodies might work well on such samples, we did not test nac-methacarn combinations further in whole planarians.
fixative choice was also critical for efficient immunofluorescence.
for most intestine-specific antibodies we tested , methacarn fixation was superior to formaldehyde fixation, especially in mg-treated animals .
in methacarn-fixed specimens, intestinal labeling was more intense, particularly in anterior regions.
additionally, non-specific background labeling was much lower as compared to formaldehyde-fixed planarians.
these results show that processing conditions  utilized to screen hybridomas do not automatically identify the “best” treatment conditions for particular antibodies.
non-intestinal mabs displayed a similar range of sensitivity to combinations of treatments .
for example, in formaldehyde-fixed samples, mab 2d <dig> labeled a subset of neuronal processes in the cephalic ganglia and ventral nerve cords, as well as a small number of cell bodies anterior and lateral to the brain .
labeling was enhanced by both mg and nac treatments, but abolished by methacarn fixation.
similarly, mab 1h <dig> preferentially labeled cns nuclei in nac-treated, formaldehyde-fixed samples, and also in mg-treated, formaldehyde-fixed samples, whether hcl or nac was used for mucus removal .
mab 2c <dig>  labeled efficiently in both methacarn and formaldehyde .
labeling of processes in the pharynx, a more internal tissue, was improved in mg-, nac-treated samples .
by contrast, mg and nac treatment reduced or eliminated labeling by mab 3g <dig>  which labeled the epidermal basement membrane only in hcl-treated, formaldehyde-fixed animals .
similarly, labeling of epidermal cell junctions by mab 3h <dig>  was also negatively affected by mg treatment, suggesting that magnesium may improve penetration of some mabs at least in part through mild histolysis of more superficial tissues.
finally, mab 2h <dig> labeled superficial body wall muscles best in nac treated samples, while labeling of internal enteric muscles surrounding intestinal branches was less sensitive to sample preparation .figure  <dig>
effects of fixation and other parameters on immunofluorescent labeling of non-intestinal tissues.
mab 2d <dig> labels a subset of cns neurons and processes after formaldehyde fixation.
mab 2c <dig> labels cell bodies around the pharynx as well as fine processes within the pharynx .
mab 3g <dig> labeling of the epidermal basement membrane  is negatively affected by magnesium treatment, methacarn fixation, and nac treatment.
mab 3h <dig> labeling of epidermal cell:cell junctions.
images taken from the posterior of the animal.
region shown is the lateral margin of the animal adjacent to the pharynx.
mab 2h <dig> labeling of body wall and enteric musculature .
sample treatment parameters are indicated at left.
in all panels, anterior is to the left.
yellow asterisks indicate conditions that yielded the most specific signal with minimal background labeling.
all samples were bleached in methanol .
scale bars:  <dig> μm ;  <dig> μm ;  <dig> μm .
taken together, these initial optimizations demonstrate that epitopes recognized by our collection of mabs are selectively sensitive to combinations of commonly used mucus removal treatments and fixatives .
furthermore, magnesium-induced relaxation is a simple step that can improve labeling of deeper tissues such as the intestine and pharynx.
optimization of these steps together is an efficient method for improving signal and reducing non-specific background labeling.table  <dig>
optimization of whole animal sample processing
mab
tissue specificity
fixation protocol
mg
mg
mg
hcl
hcl
nac
hcl
hcl
nac
mcn
fa
fa
mcn
fa
fa
qualitative assessment of signal:noise for the predominant tissue labeled by each mab.
“-“ indicates negligible or non-uniform labeling, poor morphology, and/or high background.
“+“ indicates moderate specificity, uniform labeling, and/or robust labeling accompanied by moderate background.
“++” indicates robust signal with minimal noise.
further optimization of hydrogen peroxide treatment and other sample processing steps
for many mabs, identification of the appropriate combination of magnesium treatment, mucus removal method, and fixative was sufficient to yield reproducible and specific labeling.
for several mabs, however, labeling was still inconsistent and accompanied by higher levels of background, particularly in anterior regions where secretory cells are more abundant  <cit> .
in an effort to further improve labeling, we conducted more detailed analyses of bleaching and mucus removal steps, and also tested the effects of post-fixation treatments to unmask epitopes and permeabilize tissue.
we chose three of the most inconsistent mabs  that labeled different cell types from both superficial and internal tissues for further analysis of these steps.
in order to facilitate visualization of internal tissues, hydrogen peroxide bleaching prior to immunolabeling has become a routine post-fixation step  <cit> .
bleaching also likely increases tissue permeability, allowing better penetration of molecular probes  <cit> .
in published protocols, hydrogen peroxide is usually diluted with pbs, methanol, or other diluents, but the degree to which labeling by individual antibodies is affected by diluent choice is rarely reported.
in our primary screen , hydrogen peroxide was diluted with pbs.
in subsequent optimizations , samples were bleached in hydrogen peroxide diluted with methanol.
although most antibodies were unaffected by bleaching diluent, methanol bleaching completely abolished intestinal labeling by one antibody, 3f <dig> .
consistently, no intestinal labeling was observed in methacarn-fixed animals, even when bleached in pbs, suggesting that the epitope recognized by mab 3f <dig> is sensitive to methanol .figure  <dig>
methanol bleaching abolishes labeling by mab 3f <dig>   mab 3f <dig> does not label samples bleached in methanol .
mab 3f <dig> labels the intestine in formaldehyde-fixed animals bleached in pbs .
mg treatment also substantially improves 3f <dig> labeling in hcl-treated samples, but substantially reduces labeling in nac-treated samples , demonstrating that some antibodies are uniquely sensitive to specific combinations of treatments.
in all panels, anterior is to the left.
scale bars:  <dig> μm.
to further explore diluent choice, we compared the effects of bleaching in pbs or methanol for two additional mabs.
unlike 3f <dig> labeling , 2d <dig> labeling of the cns was significantly reduced in pbs-bleached animals, particularly in more posterior regions .
2c <dig> labeling, on the other hand, was robust whether bleaching was carried out in either pbs or methanol .
thus, although many of the mabs we generated labeled planarians bleached in either pbs or methanol, side-by-side analysis indicates that bleaching diluent is an important variable that can influence optimal immunofluorescence.figure  <dig>
bleaching affects mab labeling.
planarians labeled with mab 3f <dig> after hydrogen peroxide bleaching in either pbs  or methanol .
labeling is completely abolished by methanol bleaching.
mab 2d <dig> labeling of the cns is more robust in samples bleached in methanol, particularly in posterior regions .
mab 2c <dig> labeling is unaffected by bleaching diluent.
planarians labeled with mab 3f <dig> after bleaching for the times indicated at left.
arrows  indicate signal loss in tail branches.
planarians labeled with mab 2d <dig>  arrows  indicate signal loss in tail branches.
planarians labeled with mab 2c <dig>  arrows  indicate decreased signal both within and around the pharynx.
planarians were relaxed in magnesium chloride, treated with 2% hcl  or  <dig> % nac , fixed in formaldehyde/triton x- <dig>  and bleached in 6% h2o2/pbs  or 6% h2o2/methanol .
samples were bleached for  <dig> hr in a-f. scale bars:  <dig> μm.
antibody labeling was also sensitive to the duration of bleaching.
many published protocols vaguely specify “overnight” bleaching, but systematic analysis of how bleaching time affects immunolabeling has not been reported.
again, most of our mabs adequately labeled samples bleached overnight  .
however, 3f <dig> was sensitive to both over- and under-bleaching in both hcl-treated  and nac-treated  planarians.
in addition, labeling of posterior gut branches was specifically decreased in moderately overbleached samples .
other mabs  were also sensitive to overbleaching to varying degrees .
antigen sensitivity to hydrogen peroxide treatment has been previously observed for a variety of samples from various organisms  <cit> .
our results show that although bleaching is essential for immunolabeling of whole planarians, duration of hydrogen peroxide treatment should be optimized for individual antibodies.
furthermore, caution is warranted when evaluating antibodies raised against proteins whose expression or localization is expected to vary along the anteroposterior axis.
next, we tested the effect of increasing the duration of mucus removal treatments.
for mab 3f <dig>  longer hcl and nac treatments both substantially reduced labeling .
for 2d <dig>  longer hcl treatment moderately reduced labeling in the anterior of the animal , while longer nac treatment had no effect .
for 2c <dig>  longer hcl treatment reduced labeling, as with 3f <dig> and 2d <dig> .
however, longer nac treatment increased 2c <dig> labeling within the pharynx, while simultaneously reducing labeling of peripharyngeal cell bodies and projections .
thus, longer mucus removal times generally reduce labeling intensity, but in some cases might benefit immunolabeling by increasing the accessibility of more internal organs like the pharynx.figure  <dig>
extended mucus removal time attenuates immunofluorescent labeling.
planarians labeled with mab 3f <dig> after hcl  or nac  treatment for the times indicated.
planarians labeled with mab 2d <dig>   planarians labeled with mab 2c <dig>  planarians were relaxed in magnesium chloride, treated with 2% hcl or  <dig> % nac, fixed in formaldehyde, and bleached in 6% h2o2/pbs  or 6% h2o2/meoh  .
magenta arrows indicate reduced labeling.
yellow arrow  indicates increased labeling of pharynx.
scale bars:  <dig> μm.
finally, we hypothesized that treatments to increase tissue permeability and “unmask” antigens might improve mab labeling.
we tested three methods commonly used on whole planarians: reduction, which chemically permeabilizes tissues  <cit> ; proteinase k treatment, which is thought to enzymatically cleave fixation-induced bonds  <cit> ; and heat-induced antigen retrieval , which increases antigenicity for some epitopes by a mechanism that remains incompletely understood  <cit>  .
proteinase k digestion and ar were detrimental, reducing or completely abolishing labeling for 3f <dig>  2d <dig>  and 2c <dig> .
reduction, on the other hand, improved labeling for 3f <dig> and 2d <dig>  increasing antibody penetration in anterior regions , and substantially increasing 3f <dig> signal intensity .
thus, for the three mabs we tested, reduction had the most beneficial effect.
however, since antigen-antibody interactions can respond more or less favorably to individual treatments  <cit> , we suggest that all three treatments be included in efforts to optimize antibodies with low signal, high background, or poor penetration.figure  <dig>
post-fixation treatments influence immunolabeling.
planarians labeled with mab 3f <dig> after the treatments indicated at left.
planarians labeled with mab 2d <dig>   planarians labeled with mab 2c <dig>  arrows in  indicate the prepharyngeal region that is permeabilized by reduction treatment .
asterisks indicate “best” treatment .
planarians were relaxed in magnesium chloride , treated with 2% hcl  or  <dig> % nac , fixed in formaldehyde/triton x- <dig>  and bleached in 6% h2o2/pbs  or 6% h2o2/meoh  .
reduction was conducted prior to bleaching, while proteinase k  and antigen retrieval  were conducted after bleaching.
scale bars:  <dig> μm.
mabs label planarian tissues in unbleached histological sections
histological sections provide superior spatial resolution of cellular events in some contexts  <cit> , and reduce the potential impact of issues such as antibody penetration in thick specimens.
furthermore, sections also allow easy visualization of internal tissues without requiring bleaching, an appealing option since peroxide treatment can be detrimental .
we assessed labeling by our mabs on cryosections from hcl- and nac-treated, formaldehyde-fixed planarians, and compared sections generated from both bleached and unbleached animals .
additionally, we assessed whether antigen retrieval enabled detection of epitopes on unbleached sections, since ar is widely used to unmask antigens in formaldehyde-fixed, paraffin-embedded tissue  <cit> .figure  <dig>
mab labeling of histological sections.
cryosections  from formaldehyde-fixed planarians processed as indicated.
sections labeled with secondary antibody only.
mab 3f <dig> labels the intestine .
2g <dig> labels the apical regions of intestinal cells  and epidermis.
2h <dig> labels enteric  and body wall  muscles.
schematics illustrating orientation of cross sections and tissues labeled  are to located the right of each set of panels.
all planarians were treated with hcl or nac, and fixed in formaldehyde/triton x- <dig> .
bleaching in 6% h2o2/pbs   was conducted prior to cryosectioning.
antigen retrieval was conducted after sectioning.
dorsal is to the top in all images.
conditions yielding the highest signal-to-noise for each mab are indicated with an asterisk.
scale bars:  <dig> μm.
effects of sample processing on immunolabeling of histological sections
mab
fixation protocol
hcl
nac
hcl
nac
hcl
nac
hcl
nac
hcl
nac
formaldehyde
-
bleach
ar
reduction
prot k
mab
methacarn
qualitative assessment of signal:noise for tissues labeled by selected mabs: 2g <dig>  intestine  and epidermis ; 3f <dig>  intestine; 2h <dig>  enteric muscle; 2d <dig>  cns neuropil; 2c <dig>  peripharyngeal secretory cells and projections; 3h <dig>  epidermis.
“-“ indicates negligible or non-uniform labeling, poor morphology, and/or high background.
“+“ indicates moderate specificity, uniform labeling, or strong signal accompanied by elevated non-specific labeling.
“++” indicates robust signal with minimal noise.
“n.d.”, not done.
in sections labeled with secondary antibody alone , background labeling was minimal, but higher in nac-treated sections; both bleaching and ar reduced this background significantly.
mab 3f <dig>  the antibody that was most sensitive to bleaching , labeled the intestine robustly in unbleached sections from both hcl- and nac-treated animals .
background labeling of non-intestinal tissue was higher in nac-treated sections, although much of this labeling was likely non-specific, since it also occurred in sections labeled with secondary antibody alone .
in sections from bleached animals, non-specific labeling was reduced, but intestinal signal in 3f11-labeled sections also decreased, especially in hcl-treated samples .
ar was as effective as bleaching at reducing non-specific labeling , but also abolished 3f <dig> intestinal labeling , regardless of mucus removal method.
we also co-labeled sections from hcl-treated, nac-fixed, unbleached animals with the lectin lens culinaris agglutinin , which labels intestinal goblet cells   <cit> .
overlap with 3f <dig> labeling was minimal, suggesting 3f <dig> recognizes intestinal phagocytes with a high degree of specificity.
demonstrating the variable sensitivity of antigens to processing, labeling by 2g <dig>  was affected differently by the same treatments.
first, 2g <dig> labeled the lumenal region of the intestine much more intensely in hcl-treated samples than nac-treated samples.
second, bleaching virtually eliminated 2g <dig> labeling.
third, antigen retrieval preserved intestinal labeling in hcl-treated samples, while greatly reducing non-specific mesenchymal background as well as specific epidermal signal.
one antibody, 2h <dig>  labeled both subepidermal body wall muscles  and visceral muscles surrounding intestinal branches , in patterns strikingly similar to previous studies using both phalloidin and other muscle-specific antibodies  <cit> .
further illustrating the utility of testing multiple parameters together, 2h <dig> only labeled sections after ar treatment .
interestingly, in hcl-treated sections, visceral muscle labeling was high, while external muscle labeling was low.
the opposite effect was observed in nac-treated samples, in which body wall muscles were labeled more intensely than visceral muscles.
thus, the 2h <dig> epitope may be sensitive to hcl treatment, and is degraded more quickly in external than internal tissues during mucus removal.
other antibodies displayed a similar range of treatment optima.
for example, 2d <dig> labeled neuronal projections only in unbleached, hcl-treated, non-ar sections .
2c <dig> labeled peripharyngeal secretory cells and their projections after all six treatment combinations, but signal was most specific and highest in bleached, hcl-treated sections .
3h <dig> was the least sensitive to processing, intensely labeling the epidermis in all samples .
we tested other post-fixation treatments , and also tested antibodies on sections from methacarn-fixed planarians .
as expected, mabs responded variably to these treatments.
although in no case was labeling more specific than in formaldehyde-fixed samples, ar treatment did enable muscle detection by 2h <dig> in methacarn-fixed sections , suggesting that some epitopes benefit from heat treatment, even in the absence of cross-linking fixation.
to summarize, optimal mab labeling of histological sections usually requires a specific combination of sample preparation parameters, as in whole animals.
although we initially selected for mabs that would label peroxide-bleached animals in our primary screen , we nonetheless found that on sections, labeling by a number of antibodies was completely abolished by bleaching.
one possible explanation for this result is that additional processing steps , together with the detrimental effects of peroxide bleaching, combine to degrade or alter epitopes in a way that prevents their detection.
finally, for some antibodies, ar is an effective method for reducing background and enabling antigen detection on sections from unbleached planarians.
ar is therefore a viable alternative to bleaching, since epitopes that are irreversibly damaged by peroxide treatment may be preserved in heat-treated histological sections.
a systematic approach to optimization of sample processing in planarians
we have shown that the specificity of antibodies raised against planarian tissue can be affected by every step of sample processing.
our observations are consistent with an extensive body of immunohistochemical research demonstrating that the molecular complexity of antigens makes their sensitivity to chemical treatments unique and unpredictable  <cit> .
we screened our initial hybridoma library on hcl-treated, formaldehyde-fixed, pbs-bleached planarians.
while we successfully identified antibodies that labeled under these conditions, some mabs performed better or worse when planarians were processed differently.
thus, although the axiom “you get what you screen for” holds true, screening itself does not automatically identify optimal processing conditions.
furthermore, our data imply that when screening antibodies raised against specific molecules , preparing samples several ways may be advisable.
for example, while we favor formaldehyde fixation because of its faithful preservation of protein localization and cellular morphology  <cit> , the effects of hcl or nac on specific epitopes likely need to be tested empirically.
alternatively, screening could be conducted to identify antibodies that label robustly in specific applications, for example, on samples that have first undergone in situ hybridization.
although development of a universal protocol suitable for every antibody is likely impossible, testing a limited number of methods together can efficiently identify conditions that preserve antigenicity and achieve excellent signal-to-noise for many antibodies .
for whole planarians, we have developed a two stage optimization workflow .
in the first stage , mucolytic agents, fixatives, and bleaching diluents are tested in combination for a total of six initial conditions.
although we did not extensively test methacarn together with milder nac treatment, such a combination could be included, increasing the total number of initial conditions to eight.
in an optional second stage , other parameters such as magnesium-induced relaxation, bleaching time, antigen retrieval, and antibody dilution can be tested to further refine a protocol for a particular antibody, as we have done for mabs 3f <dig>  2d <dig>  and 2c <dig>  alternative approaches are also possible.
for example, reduction and proteinase k treatment could be tested during the first round, increasing the number of initial combinations to  <dig> figure  <dig>
a combinatorial method for optimization of antibody labeling in planarians.
schematic of first round optimization steps for whole animals.
schematic of potential further optimization steps.
optimization steps on cryosections.
examples of optimized protocols for antibodies raised in this study.
because peroxide bleaching affects some antigens detrimentally, and because some antigens may require unmasking, we also recommend testing mabs on cryosections from bleached and unbleached planarians in combination with antigen retrieval .
we routinely adhere sections from planarians prepared four different ways to the same slide.
using two slides, eight combinations including antigen retrieval can be analyzed simultaneously in one experiment.
again, parameters can be easily substituted; for example, bleached samples could be omitted in favor of samples fixed in methacarn, or reduction could be tested instead of antigen retrieval.
by approaching sample preparation systematically, we have substantially improved processing protocols for several of the monoclonal antibodies we generated .
these protocols are suitable for use on uninjured animals as well as regenerates, and enable visualization of intestinal remodeling and growth , re-establishment of the central nervous system after head amputation , and the de novo development of secretory cells and their projections into the pharynx .
we have assembled step-by-step protocols detailing each stage of sample preparation, including examples of initial optimization steps for whole animals, as well as summary protocols for several individual antibodies .figure  <dig>
mabs label regenerating intestine, secretory cells, and central nervous system in specimens processed using optimized protocols.
3f <dig> labels the intestine in tail fragments regenerating a new head, including anterior regions that remodel  and elongate  at early and intermediate stages of intestinal regeneration, respectively.
after head amputation, 2d <dig> labels the regenerating brain, including new neuronal projections , and cell bodies .
2c <dig> labels the regenerating peripharyngeal secretory system in tail fragments, including the first appearance of cell bodies  <dig> days after amputation , and projections within the pharynx  <dig> days after amputation .
days after amputation are indicated at left.
anterior is to the left in all panels.
dotted magenta lines indicate approximate boundary between new tissue  and old tissue .
all samples were processed using the optimized protocol for each mab described in additional file  <dig>  scale bars:  <dig> μm ;  <dig> μm .
future directions
although we evaluated many of the more commonly used fixatives and sample preparation conditions, our analysis was not exhaustive.
alternative fixatives, mucus removal treatments, bleaching agents, and tissue permeabilization methods remain to be tested or might emerge in the future.
for example, a combined relaxing agent and fixative has been used previously to prepare planarians for paraffin embedding, sectioning, and immunolabeling  <cit> .
the modular nature of our optimization workflow allows straightforward substitution or addition of such treatments.
additionally, although we conducted tests of the duration of bleaching and mucus removal, we have not rigorously explored treatment time as a parameter.
in particular, fixation and permeabilization may need to be extended for larger planarians, as noted for in situ hybridization protocols  <cit> .
similarly, initial trials suggest that small animals and tissue fragments  may be more sensitive to hydrogen peroxide treatment, requiring bleaching for  <dig> hr or less, at least for some antibodies.
CONCLUSIONS
we raised a panel of mabs against planarian intestinal cells, and identified optimal sample preparation conditions for several antibodies.
serendipitously, our screen also yielded markers for non-intestinal cell types, reagents that may benefit studies of regeneration of the nervous system, pharynx, and epidermis.
our results reinforce the feasibility of producing tissue-specific markers using whole planarian cells as an immunogen.
furthermore, our approach to testing multiple parameters together during sample processing should accelerate future efforts to develop planarian-specific antibodies, and to extend investigation of regenerative mechanisms to post-transcriptional aspects of gene expression including protein localization and modification.
finally, our observations emphasize the long-appreciated sensitivity of antibody specificity to fixation and other treatments, and the utility of systematically testing multiple approaches when addressing organism-specific idiosyncracies such as mucus secretions or tissue permeability.
our experiences and approaches may facilitate efforts to develop immunological resources in other emerging model organisms.

 interest in the cellular and molecular mechanisms of regeneration has stimulated a resurgence of investigations in a growing cohort of organisms. for example, planarians can recover from nearly any plane of transection, re-establishing their body plan and rebuilding internal organs such as the nervous and digestive systems within a week after injury. recent investigations have illuminated some of the mechanisms of planarian regeneration, including the re-establishment of axial polarity, somatic stem cell dynamics, tissue remodeling, organogenesis, reproductive maturation and germ cell development, and the molecular nature of regenerative competence. as in many experimental animal models, these advances have required the adaptation and development of a range of tools and techniques, including methods for visualizing specific organs, tissues, and cell types. in particular, optimization of protocols for in situ hybridization and sample processing for electron microscopy have dramatically increased the resolution of regenerative events at the cellular level. by contrast, a rigorous analysis of the influence of specific steps during sample preparation for immunofluorescence has not been undertaken, and the collection of planarian-specific antibodies remains limited. development of more systematic approaches for testing the effects of specific parameters on immunolabeling by individual antibodies would accelerate the generation of cell- and tissue-specific reagents, and expedite studies of post-transcriptional aspects of gene expression during regeneration, such as protein localization and modification. historically, characterization of planarian tissues and studies of their responses to injury were conducted using histological stains, vital dyes, and electron microscopy. more recently, significant improvements to in situ hybridization protocols have enabled the use of rna probes to label organs, subpopulations of cells, and ribonucleoprotein particles. in addition to these methods, protocols utilizing both lectins and antibodies as cell-specific probes have also been developed. these protocols are less labor intensive and more economical than ish protocols, and, in addition to detection of specific cell types, enable resolution of subcellular regions such as membranes, nuclei, and neuronal processes. a variety of lectins and antibodies have been generated or identified that label the secretory system, reproductive system, nervous system, intestine, protonephridia, muscles, and stem cells. in addition, commercial and publicly available antibodies that cross-react with planarian antigens in the nervous system and other tissues have been identified. although antibodies are most often raised against specific molecules, monoclonal antibodies have also been generated in large-scale screens using purified cells, tissues, or whole-animal homogenates as immunogens. such screens have yielded specific markers for neurons and their projections, regenerating tissues, and other cell types in a variety of organisms. tissue-based mab screens bypass potential difficulties such as the need to identify highly expressed proteins or immunogenic regions appropriate for production of fusion proteins. additionally, mab screens result in the generation of clonal, immortal hybridoma lines and therefore a theoretically inexhaustible supply of antibody. several mab screens have been conducted using planarian cells and extracts, and have yielded markers for various tissues and cell types. despite recent progress, the repertoire of antibodies that recognize planarian tissues is still limited, and a greater collection of reagents that label cell types unique to specific organ systems is needed. for example, regeneration of the intestine requires both remodeling of pre-existing, post-mitotic intestinal tissue and addition of new intestinal cells at the growing ends of regenerating gut branches. antibodies currently used to label the intestine lack specificity, labeling additional tissues such as pharynx, epidermis, and nervous system. furthermore, some of these antibodies label only subregions of intestinal cells such as the apical surface, making them less than ideal for analysis of remodeling and growth during intestinal morphogenesis. in order to develop more specific intestinal antibodies, we took advantage of a protocol we recently developed that enables purification of intestinal cells, and conducted a mab screen using these purified cells as immunogens. we generated ten mabs that labeled the intestine, as well as mabs that label the nervous system, epidermis, secretory cells, and other cell types. because sample processing is known to influence antibody-antigen interactions, we also systematically evaluated the effects of various parameters during fixation of planarian tissue, including chemical treatments to remove mucus and pigmentation. this analysis led to the identification of optimal sample preparation protocols for several mabs, and to the development of an optimization workflow that efficiently tests the influence of multiple processing steps on immunolabeling in planarians.  a monoclonal antibody screen utilizing purified intestinal phagocytes as immunogens the planarian intestinal epithelium is comprised of two cell types: secretory goblet cells that release digestive enzymes after food ingestion, and absorptive phagocytes that engulf food for intracellular digestion. intestinal phagocytes retain dyes, beads, and other compounds for up to several weeks after they are ingested. we recently developed a protocol in which animals are fed iron beads and dissociated into single cell suspensions, enabling the purification of phagocytes by magnetic sorting. using this protocol, we collected phagocytes from more than planarians. after fixation, these cells were used as immunogens for a monoclonal antibody screen.figure generation of monoclonal antibodies that recognize the planarian intestine and other tissues. screen strategy. preimmunization serum did not detectably label planarian tissue. test sera ubiquitously labeled planarian tissues, with slightly elevated intestinal labeling. pie chart depicting the number of clones in the primary screen with indicated labeling specificities. examples and schematics of immunolabeling from the primary screen. mabs that label the intestine. mabs that label the nervous system. examples of mabs that label other non-intestinal tissues. anterior is to the top in all images. cell and tissue types are indicated in each panel. “cns”, central nervous system. “vnc”, ventral nerve cord. scale bars: μm; μm; μm. three mice were immunized; test bleeds from all three mice labeled fixed planarians, while preimmune sera did not. immunofluorescence was ubiquitous, likely reflecting the fact that many intestinal antigens are also expressed in other planarian cell types. we also observed slightly elevated signal in the intestine for all three test sera. one mouse was chosen for final immunization and hybridoma generation. after fusion of splenocytes with myeloma cells and cloning of the resulting hybridomas, supernatants from primary lines were screened for immunoreactivity with fixed planarians. in total, supernatants labeled planarian tissues at levels visibly higher than secondary antibody controls. seventy supernatants labeled the intestine. in addition, supernatants labeled non-intestinal tissues. these included pigment cells of the optic cup, neurons and their processes, muscles, epidermis, secretory cells, and cilia. twenty-four antibodies that labeled non-intestinal tissues also labeled the intestine, suggesting the recognition of highly immunogenic antigens that are expressed at lower levels in the intestine than in other tissues. alternatively, some non-intestinal antibodies might also have been generated against antigens expressed by contaminating cells or cell fragments in intestinal cell preparations. in previous monoclonal screens, less than 8% of antibody-producing clones labeled intestinal tissue. by contrast, in our screen ~25% of positive clones labeled the intestine with marked specificity. because intestinal cells comprise only 3-8% of all planarian cell types, these results show that immunization with an enriched cell population is an efficient strategy for generating cell type-specific mabs. we retained hybridomas, giving preference to those that produced antibodies labeling the intestine and other specific cell types with high signal and low background. of hybridoma lines produced igm class antibodies, while two lines produced igg mabs. igm-producing hybridomas can dominate an immune response due to underimmunization with suboptimal amounts of antigen, or an abundance of complex carbohydrates and lipids when whole cells are used as an immunogen. consistently, a recent effort to generate mabs against membranes purified from planarian stem cells yielded many igm-producing lines. although igm mabs sometimes have lower affinity, reduced specificity, and decreased ability to penetrate tissue, our screening strategy identified igm mabs that labeled specific cell types and penetrated tissues well. each of the hybridomas was subcloned, and we identified at least one unique line that continued to secrete high levels of specific mab after rescreening, freezing, and expansion. subclones were deposited in the developmental studies hybridoma bank for archival and public distribution.table monoclonal antibodies, isotypes, and tissue specificity clone isotype* tissue recognized *all mabs possessed kappa light chains. †pattern based on primary screen; mab has not been characterized further. optimization of planarian relaxation, mucus removal, and fixation improves immunolabeling although our mabs consistently labeled specific tissues in the primary screen, levels of both signal and non-specific background often varied from animal to animal and within different regions of individual animals. because epitope accessibility and antibody specificity depend critically on fixative choice and other sample processing steps, we hypothesized that optimization of fixation and other treatments would improve labeling with some mabs. processing of planarian tissue for immunofluorescence requires several steps in addition to fixation, including chemical removal of mucus secretions prior to fixation, bleaching to remove pigmentation and enable visualization of internal structures, and post-fixation treatments to increase tissue permeability and expose antigens. a range of approaches to these steps have been developed to prepare planarians for whole mount in situ hybridization, immunolabeling, and histological analysis. in our primary screen, we tested hybridoma supernatants on hcl-treated, formaldehyde-fixed planarians that were bleached in hydrogen peroxide diluted in pbs. because formal demonstrations of the effects of specific treatments at each stage of sample processing are rarely reported, we evaluated additional approaches to these steps simultaneously by testing combinations of treatments together.figure sample processing influences immunofluorescent labeling of the planarian intestine. overview of steps during fixation and processing. mab 4d labeling. mab 3g mab 2g for all three mabs, labeling was best in animals relaxed in magnesium and fixed with methacarn. in some samples, magnesium treatment also improved labeling of anterior intestinal branches. “mg”, magnesium-induced relaxation. “nac”, n-acetyl-l-cysteine treatment. “hcl”, hcl treatment. “fa”, formaldehyde/triton x- fixation. “mcn”, methacarn fixation. “tsa”, tyramide signal amplification. samples were bleached in methanol/6% h2o. scale bars: μm. we began by comparing two chemical treatments commonly used to relax planarians and remove their mucus secretions prior to fixation: hydrochloric acid and n-acetyl-l-cysteine. we also compared formaldehyde, a cross-linking fixative, with methacarn, a coagulating fixative, reasoning that antibodies raised against cells fixed in formaldehyde and methanol would be most likely to react with planarian tissues that had been fixed similarly. additionally, we tested magnesium chloride, which has been used to relax marine and freshwater invertebrates prior to fixation, as an extra step prior to mucus removal. we have previously utilized magnesium-induced relaxation to increase resolution of individual branches of the planarian intestine. for these optimizations, we utilized indirect detection, which dramatically improved signal intensity for supernatants generated after the primary screen. furthermore, blocking time was increased to overnight, a step that moderately improved signal-to-noise for some intestinal antibodies, without affecting labeling efficiency for others. for intestine-specific mabs, magnesium treatment universally improved labeling. for example, in the absence of magnesium treatment, mabs 4d and 3g labeled anterior and posterior intestinal branches in hcl-treated, formaldehyde-fixed planarians, but penetration in the prepharyngeal region was poor. when animals were relaxed in magnesium prior to hcl treatment, labeling of the primary anterior intestinal branch was dramatically improved. similar results were observed for a third mab, 2g in methacarn-fixed planarians. mucus removal method also affected intestinal labeling, albeit more moderately. for example, hcl treatment improved signal and reduced non-specific labeling by antibodies 4d and 3g in formaldehyde-fixed planarians, while nac treatment led to slightly more specific labeling by 2g. we also attempted to fix nac-treated animals with methacarn, but animals disintegrated during bleaching. milder nac treatments or reduced h2o concentration during bleaching improved integrity, but abolished intestinal labeling. although we cannot rule out that some antibodies might work well on such samples, we did not test nac-methacarn combinations further in whole planarians. fixative choice was also critical for efficient immunofluorescence. for most intestine-specific antibodies we tested, methacarn fixation was superior to formaldehyde fixation, especially in mg-treated animals. in methacarn-fixed specimens, intestinal labeling was more intense, particularly in anterior regions. additionally, non-specific background labeling was much lower as compared to formaldehyde-fixed planarians. these results show that processing conditions utilized to screen hybridomas do not automatically identify the “best” treatment conditions for particular antibodies. non-intestinal mabs displayed a similar range of sensitivity to combinations of treatments. for example, in formaldehyde-fixed samples, mab 2d labeled a subset of neuronal processes in the cephalic ganglia and ventral nerve cords, as well as a small number of cell bodies anterior and lateral to the brain. labeling was enhanced by both mg and nac treatments, but abolished by methacarn fixation. similarly, mab 1h preferentially labeled cns nuclei in nac-treated, formaldehyde-fixed samples, and also in mg-treated, formaldehyde-fixed samples, whether hcl or nac was used for mucus removal. mab 2c labeled efficiently in both methacarn and formaldehyde. labeling of processes in the pharynx, a more internal tissue, was improved in mg-, nac-treated samples. by contrast, mg and nac treatment reduced or eliminated labeling by mab 3g which labeled the epidermal basement membrane only in hcl-treated, formaldehyde-fixed animals. similarly, labeling of epidermal cell junctions by mab 3h was also negatively affected by mg treatment, suggesting that magnesium may improve penetration of some mabs at least in part through mild histolysis of more superficial tissues. finally, mab 2h labeled superficial body wall muscles best in nac treated samples, while labeling of internal enteric muscles surrounding intestinal branches was less sensitive to sample preparation.figure effects of fixation and other parameters on immunofluorescent labeling of non-intestinal tissues. mab 2d labels a subset of cns neurons and processes after formaldehyde fixation. mab 2c labels cell bodies around the pharynx as well as fine processes within the pharynx. mab 3g labeling of the epidermal basement membrane is negatively affected by magnesium treatment, methacarn fixation, and nac treatment. mab 3h labeling of epidermal cell:cell junctions. images taken from the posterior of the animal. region shown is the lateral margin of the animal adjacent to the pharynx. mab 2h labeling of body wall and enteric musculature. sample treatment parameters are indicated at left. in all panels, anterior is to the left. yellow asterisks indicate conditions that yielded the most specific signal with minimal background labeling. all samples were bleached in methanol. scale bars: μm; μm; μm. taken together, these initial optimizations demonstrate that epitopes recognized by our collection of mabs are selectively sensitive to combinations of commonly used mucus removal treatments and fixatives. furthermore, magnesium-induced relaxation is a simple step that can improve labeling of deeper tissues such as the intestine and pharynx. optimization of these steps together is an efficient method for improving signal and reducing non-specific background labeling.table optimization of whole animal sample processing mab tissue specificity fixation protocol mg mg mg hcl hcl nac hcl hcl nac mcn fa fa mcn fa fa qualitative assessment of signal:noise for the predominant tissue labeled by each mab. “-“ indicates negligible or non-uniform labeling, poor morphology, and/or high background. “+“ indicates moderate specificity, uniform labeling, and/or robust labeling accompanied by moderate background. “++” indicates robust signal with minimal noise. further optimization of hydrogen peroxide treatment and other sample processing steps for many mabs, identification of the appropriate combination of magnesium treatment, mucus removal method, and fixative was sufficient to yield reproducible and specific labeling. for several mabs, however, labeling was still inconsistent and accompanied by higher levels of background, particularly in anterior regions where secretory cells are more abundant. in an effort to further improve labeling, we conducted more detailed analyses of bleaching and mucus removal steps, and also tested the effects of post-fixation treatments to unmask epitopes and permeabilize tissue. we chose three of the most inconsistent mabs that labeled different cell types from both superficial and internal tissues for further analysis of these steps. in order to facilitate visualization of internal tissues, hydrogen peroxide bleaching prior to immunolabeling has become a routine post-fixation step. bleaching also likely increases tissue permeability, allowing better penetration of molecular probes. in published protocols, hydrogen peroxide is usually diluted with pbs, methanol, or other diluents, but the degree to which labeling by individual antibodies is affected by diluent choice is rarely reported. in our primary screen, hydrogen peroxide was diluted with pbs. in subsequent optimizations, samples were bleached in hydrogen peroxide diluted with methanol. although most antibodies were unaffected by bleaching diluent, methanol bleaching completely abolished intestinal labeling by one antibody, 3f. consistently, no intestinal labeling was observed in methacarn-fixed animals, even when bleached in pbs, suggesting that the epitope recognized by mab 3f is sensitive to methanol.figure methanol bleaching abolishes labeling by mab 3f mab 3f does not label samples bleached in methanol. mab 3f labels the intestine in formaldehyde-fixed animals bleached in pbs. mg treatment also substantially improves 3f labeling in hcl-treated samples, but substantially reduces labeling in nac-treated samples, demonstrating that some antibodies are uniquely sensitive to specific combinations of treatments. in all panels, anterior is to the left. scale bars: μm. to further explore diluent choice, we compared the effects of bleaching in pbs or methanol for two additional mabs. unlike 3f labeling, 2d labeling of the cns was significantly reduced in pbs-bleached animals, particularly in more posterior regions. 2c labeling, on the other hand, was robust whether bleaching was carried out in either pbs or methanol. thus, although many of the mabs we generated labeled planarians bleached in either pbs or methanol, side-by-side analysis indicates that bleaching diluent is an important variable that can influence optimal immunofluorescence.figure bleaching affects mab labeling. planarians labeled with mab 3f after hydrogen peroxide bleaching in either pbs or methanol. labeling is completely abolished by methanol bleaching. mab 2d labeling of the cns is more robust in samples bleached in methanol, particularly in posterior regions. mab 2c labeling is unaffected by bleaching diluent. planarians labeled with mab 3f after bleaching for the times indicated at left. arrows indicate signal loss in tail branches. planarians labeled with mab 2d arrows indicate signal loss in tail branches. planarians labeled with mab 2c arrows indicate decreased signal both within and around the pharynx. planarians were relaxed in magnesium chloride, treated with 2% hcl or % nac, fixed in formaldehyde/triton x- and bleached in 6% h2o2/pbs or 6% h2o2/methanol. samples were bleached for hr in a-f. scale bars: μm. antibody labeling was also sensitive to the duration of bleaching. many published protocols vaguely specify “overnight” bleaching, but systematic analysis of how bleaching time affects immunolabeling has not been reported. again, most of our mabs adequately labeled samples bleached overnight. however, 3f was sensitive to both over- and under-bleaching in both hcl-treated and nac-treated planarians. in addition, labeling of posterior gut branches was specifically decreased in moderately overbleached samples. other mabs were also sensitive to overbleaching to varying degrees. antigen sensitivity to hydrogen peroxide treatment has been previously observed for a variety of samples from various organisms. our results show that although bleaching is essential for immunolabeling of whole planarians, duration of hydrogen peroxide treatment should be optimized for individual antibodies. furthermore, caution is warranted when evaluating antibodies raised against proteins whose expression or localization is expected to vary along the anteroposterior axis. next, we tested the effect of increasing the duration of mucus removal treatments. for mab 3f longer hcl and nac treatments both substantially reduced labeling. for 2d longer hcl treatment moderately reduced labeling in the anterior of the animal, while longer nac treatment had no effect. for 2c longer hcl treatment reduced labeling, as with 3f and 2d. however, longer nac treatment increased 2c labeling within the pharynx, while simultaneously reducing labeling of peripharyngeal cell bodies and projections. thus, longer mucus removal times generally reduce labeling intensity, but in some cases might benefit immunolabeling by increasing the accessibility of more internal organs like the pharynx.figure extended mucus removal time attenuates immunofluorescent labeling. planarians labeled with mab 3f after hcl or nac treatment for the times indicated. planarians labeled with mab 2d planarians labeled with mab 2c planarians were relaxed in magnesium chloride, treated with 2% hcl or % nac, fixed in formaldehyde, and bleached in 6% h2o2/pbs or 6% h2o2/meoh. magenta arrows indicate reduced labeling. yellow arrow indicates increased labeling of pharynx. scale bars: μm. finally, we hypothesized that treatments to increase tissue permeability and “unmask” antigens might improve mab labeling. we tested three methods commonly used on whole planarians: reduction, which chemically permeabilizes tissues; proteinase k treatment, which is thought to enzymatically cleave fixation-induced bonds; and heat-induced antigen retrieval, which increases antigenicity for some epitopes by a mechanism that remains incompletely understood. proteinase k digestion and ar were detrimental, reducing or completely abolishing labeling for 3f 2d and 2c. reduction, on the other hand, improved labeling for 3f and 2d increasing antibody penetration in anterior regions, and substantially increasing 3f signal intensity. thus, for the three mabs we tested, reduction had the most beneficial effect. however, since antigen-antibody interactions can respond more or less favorably to individual treatments, we suggest that all three treatments be included in efforts to optimize antibodies with low signal, high background, or poor penetration.figure post-fixation treatments influence immunolabeling. planarians labeled with mab 3f after the treatments indicated at left. planarians labeled with mab 2d planarians labeled with mab 2c arrows in indicate the prepharyngeal region that is permeabilized by reduction treatment. asterisks indicate “best” treatment. planarians were relaxed in magnesium chloride, treated with 2% hcl or % nac, fixed in formaldehyde/triton x- and bleached in 6% h2o2/pbs or 6% h2o2/meoh. reduction was conducted prior to bleaching, while proteinase k and antigen retrieval were conducted after bleaching. scale bars: μm. mabs label planarian tissues in unbleached histological sections histological sections provide superior spatial resolution of cellular events in some contexts, and reduce the potential impact of issues such as antibody penetration in thick specimens. furthermore, sections also allow easy visualization of internal tissues without requiring bleaching, an appealing option since peroxide treatment can be detrimental. we assessed labeling by our mabs on cryosections from hcl- and nac-treated, formaldehyde-fixed planarians, and compared sections generated from both bleached and unbleached animals. additionally, we assessed whether antigen retrieval enabled detection of epitopes on unbleached sections, since ar is widely used to unmask antigens in formaldehyde-fixed, paraffin-embedded tissue.figure mab labeling of histological sections. cryosections from formaldehyde-fixed planarians processed as indicated. sections labeled with secondary antibody only. mab 3f labels the intestine. 2g labels the apical regions of intestinal cells and epidermis. 2h labels enteric and body wall muscles. schematics illustrating orientation of cross sections and tissues labeled are to located the right of each set of panels. all planarians were treated with hcl or nac, and fixed in formaldehyde/triton x-. bleaching in 6% h2o2/pbs was conducted prior to cryosectioning. antigen retrieval was conducted after sectioning. dorsal is to the top in all images. conditions yielding the highest signal-to-noise for each mab are indicated with an asterisk. scale bars: μm. effects of sample processing on immunolabeling of histological sections mab fixation protocol hcl nac hcl nac hcl nac hcl nac hcl nac formaldehyde - bleach ar reduction prot k mab methacarn qualitative assessment of signal:noise for tissues labeled by selected mabs: 2g intestine and epidermis; 3f intestine; 2h enteric muscle; 2d cns neuropil; 2c peripharyngeal secretory cells and projections; 3h epidermis. “-“ indicates negligible or non-uniform labeling, poor morphology, and/or high background. “+“ indicates moderate specificity, uniform labeling, or strong signal accompanied by elevated non-specific labeling. “++” indicates robust signal with minimal noise. “n.d.”, not done. in sections labeled with secondary antibody alone, background labeling was minimal, but higher in nac-treated sections; both bleaching and ar reduced this background significantly. mab 3f the antibody that was most sensitive to bleaching, labeled the intestine robustly in unbleached sections from both hcl- and nac-treated animals. background labeling of non-intestinal tissue was higher in nac-treated sections, although much of this labeling was likely non-specific, since it also occurred in sections labeled with secondary antibody alone. in sections from bleached animals, non-specific labeling was reduced, but intestinal signal in 3f11-labeled sections also decreased, especially in hcl-treated samples. ar was as effective as bleaching at reducing non-specific labeling, but also abolished 3f intestinal labeling, regardless of mucus removal method. we also co-labeled sections from hcl-treated, nac-fixed, unbleached animals with the lectin lens culinaris agglutinin, which labels intestinal goblet cells. overlap with 3f labeling was minimal, suggesting 3f recognizes intestinal phagocytes with a high degree of specificity. demonstrating the variable sensitivity of antigens to processing, labeling by 2g was affected differently by the same treatments. first, 2g labeled the lumenal region of the intestine much more intensely in hcl-treated samples than nac-treated samples. second, bleaching virtually eliminated 2g labeling. third, antigen retrieval preserved intestinal labeling in hcl-treated samples, while greatly reducing non-specific mesenchymal background as well as specific epidermal signal. one antibody, 2h labeled both subepidermal body wall muscles and visceral muscles surrounding intestinal branches, in patterns strikingly similar to previous studies using both phalloidin and other muscle-specific antibodies. further illustrating the utility of testing multiple parameters together, 2h only labeled sections after ar treatment. interestingly, in hcl-treated sections, visceral muscle labeling was high, while external muscle labeling was low. the opposite effect was observed in nac-treated samples, in which body wall muscles were labeled more intensely than visceral muscles. thus, the 2h epitope may be sensitive to hcl treatment, and is degraded more quickly in external than internal tissues during mucus removal. other antibodies displayed a similar range of treatment optima. for example, 2d labeled neuronal projections only in unbleached, hcl-treated, non-ar sections. 2c labeled peripharyngeal secretory cells and their projections after all six treatment combinations, but signal was most specific and highest in bleached, hcl-treated sections. 3h was the least sensitive to processing, intensely labeling the epidermis in all samples. we tested other post-fixation treatments, and also tested antibodies on sections from methacarn-fixed planarians. as expected, mabs responded variably to these treatments. although in no case was labeling more specific than in formaldehyde-fixed samples, ar treatment did enable muscle detection by 2h in methacarn-fixed sections, suggesting that some epitopes benefit from heat treatment, even in the absence of cross-linking fixation. to summarize, optimal mab labeling of histological sections usually requires a specific combination of sample preparation parameters, as in whole animals. although we initially selected for mabs that would label peroxide-bleached animals in our primary screen, we nonetheless found that on sections, labeling by a number of antibodies was completely abolished by bleaching. one possible explanation for this result is that additional processing steps, together with the detrimental effects of peroxide bleaching, combine to degrade or alter epitopes in a way that prevents their detection. finally, for some antibodies, ar is an effective method for reducing background and enabling antigen detection on sections from unbleached planarians. ar is therefore a viable alternative to bleaching, since epitopes that are irreversibly damaged by peroxide treatment may be preserved in heat-treated histological sections. a systematic approach to optimization of sample processing in planarians we have shown that the specificity of antibodies raised against planarian tissue can be affected by every step of sample processing. our observations are consistent with an extensive body of immunohistochemical research demonstrating that the molecular complexity of antigens makes their sensitivity to chemical treatments unique and unpredictable. we screened our initial hybridoma library on hcl-treated, formaldehyde-fixed, pbs-bleached planarians. while we successfully identified antibodies that labeled under these conditions, some mabs performed better or worse when planarians were processed differently. thus, although the axiom “you get what you screen for” holds true, screening itself does not automatically identify optimal processing conditions. furthermore, our data imply that when screening antibodies raised against specific molecules, preparing samples several ways may be advisable. for example, while we favor formaldehyde fixation because of its faithful preservation of protein localization and cellular morphology, the effects of hcl or nac on specific epitopes likely need to be tested empirically. alternatively, screening could be conducted to identify antibodies that label robustly in specific applications, for example, on samples that have first undergone in situ hybridization. although development of a universal protocol suitable for every antibody is likely impossible, testing a limited number of methods together can efficiently identify conditions that preserve antigenicity and achieve excellent signal-to-noise for many antibodies. for whole planarians, we have developed a two stage optimization workflow. in the first stage, mucolytic agents, fixatives, and bleaching diluents are tested in combination for a total of six initial conditions. although we did not extensively test methacarn together with milder nac treatment, such a combination could be included, increasing the total number of initial conditions to eight. in an optional second stage, other parameters such as magnesium-induced relaxation, bleaching time, antigen retrieval, and antibody dilution can be tested to further refine a protocol for a particular antibody, as we have done for mabs 3f 2d and 2c alternative approaches are also possible. for example, reduction and proteinase k treatment could be tested during the first round, increasing the number of initial combinations to figure a combinatorial method for optimization of antibody labeling in planarians. schematic of first round optimization steps for whole animals. schematic of potential further optimization steps. optimization steps on cryosections. examples of optimized protocols for antibodies raised in this study. because peroxide bleaching affects some antigens detrimentally, and because some antigens may require unmasking, we also recommend testing mabs on cryosections from bleached and unbleached planarians in combination with antigen retrieval. we routinely adhere sections from planarians prepared four different ways to the same slide. using two slides, eight combinations including antigen retrieval can be analyzed simultaneously in one experiment. again, parameters can be easily substituted; for example, bleached samples could be omitted in favor of samples fixed in methacarn, or reduction could be tested instead of antigen retrieval. by approaching sample preparation systematically, we have substantially improved processing protocols for several of the monoclonal antibodies we generated. these protocols are suitable for use on uninjured animals as well as regenerates, and enable visualization of intestinal remodeling and growth, re-establishment of the central nervous system after head amputation, and the de novo development of secretory cells and their projections into the pharynx. we have assembled step-by-step protocols detailing each stage of sample preparation, including examples of initial optimization steps for whole animals, as well as summary protocols for several individual antibodies.figure mabs label regenerating intestine, secretory cells, and central nervous system in specimens processed using optimized protocols. 3f labels the intestine in tail fragments regenerating a new head, including anterior regions that remodel and elongate at early and intermediate stages of intestinal regeneration, respectively. after head amputation, 2d labels the regenerating brain, including new neuronal projections, and cell bodies. 2c labels the regenerating peripharyngeal secretory system in tail fragments, including the first appearance of cell bodies days after amputation, and projections within the pharynx days after amputation. days after amputation are indicated at left. anterior is to the left in all panels. dotted magenta lines indicate approximate boundary between new tissue and old tissue. all samples were processed using the optimized protocol for each mab described in additional file scale bars: μm; μm. future directions although we evaluated many of the more commonly used fixatives and sample preparation conditions, our analysis was not exhaustive. alternative fixatives, mucus removal treatments, bleaching agents, and tissue permeabilization methods remain to be tested or might emerge in the future. for example, a combined relaxing agent and fixative has been used previously to prepare planarians for paraffin embedding, sectioning, and immunolabeling. the modular nature of our optimization workflow allows straightforward substitution or addition of such treatments. additionally, although we conducted tests of the duration of bleaching and mucus removal, we have not rigorously explored treatment time as a parameter. in particular, fixation and permeabilization may need to be extended for larger planarians, as noted for in situ hybridization protocols. similarly, initial trials suggest that small animals and tissue fragments may be more sensitive to hydrogen peroxide treatment, requiring bleaching for hr or less, at least for some antibodies.  we raised a panel of mabs against planarian intestinal cells, and identified optimal sample preparation conditions for several antibodies. serendipitously, our screen also yielded markers for non-intestinal cell types, reagents that may benefit studies of regeneration of the nervous system, pharynx, and epidermis. our results reinforce the feasibility of producing tissue-specific markers using whole planarian cells as an immunogen. furthermore, our approach to testing multiple parameters together during sample processing should accelerate future efforts to develop planarian-specific antibodies, and to extend investigation of regenerative mechanisms to post-transcriptional aspects of gene expression including protein localization and modification. finally, our observations emphasize the long-appreciated sensitivity of antibody specificity to fixation and other treatments, and the utility of systematically testing multiple approaches when addressing organism-specific idiosyncracies such as mucus secretions or tissue permeability. our experiences and approaches may facilitate efforts to develop immunological resources in other emerging model organisms. 
we also developed a protocol for optimizing specimen processing that will accelerate future efforts to generate planarian-specific antibodies, and to extend functional genetic studies of regeneration to post-transcriptional aspects of gene expression, such as protein localization or modification. we found that fixative choice, treatments to remove mucus and bleach pigment, as well as methods for tissue permeabilization and antigen retrieval profoundly influenced labeling by individual antibodies. these experiments led to the development of a step-by-step workflow for determining optimal specimen preparation for labeling whole planarians as well as unbleached histological sections. we generated a collection of monoclonal antibodies recognizing the planarian intestine and other tissues; these antibodies will facilitate studies of planarian tissue morphogenesis. in order to improve signal intensity and reduce non-specific background for a subset of mabs, we evaluated the effects of fixation and other steps during sample processing. for example, in the planarian schmidtea mediterranea, substantial improvements to nucleic acid hybridization and electron microscopy protocols have facilitated the visualization of regenerative events at the cellular level. efforts to elucidate the cellular and molecular mechanisms of regeneration have required the application of methods to detect specific cell types and tissues in a growing cohort of experimental animal models. this approach yielded ten antibodies that recognized intestinal epitopes, as well as markers for the central nervous system, musculature, secretory cells, and epidermis. by contrast, immunological resources have been slower to emerge. our efforts demonstrate the importance of systematically testing multiple approaches to species-specific idiosyncracies, such as mucus removal and pigment bleaching, and may serve as a template for the development of immunological resources in other emerging model organisms. planarianregenerationintestinemonoclonal antibody screenimmunohistochemistryimmunofluorescenceissue-copyright-statement© the author 2014 specifically, the repertoire of antibodies recognizing planarian antigens remains limited, and a more systematic approach is needed to evaluate the effects of processing steps required during sample preparation for immunolabeling.
 efforts to elucidate the cellular and molecular mechanisms of regeneration have required the application of methods to detect specific cell types and tissues in a growing cohort of experimental animal models. for example, in the planarian schmidtea mediterranea, substantial improvements to nucleic acid hybridization and electron microscopy protocols have facilitated the visualization of regenerative events at the cellular level. by contrast, immunological resources have been slower to emerge. specifically, the repertoire of antibodies recognizing planarian antigens remains limited, and a more systematic approach is needed to evaluate the effects of processing steps required during sample preparation for immunolabeling.  to address these issues and to facilitate studies of planarian digestive system regeneration, we conducted a monoclonal antibody screen using phagocytic intestinal cells purified from the digestive tracts of living planarians as immunogens. this approach yielded ten antibodies that recognized intestinal epitopes, as well as markers for the central nervous system, musculature, secretory cells, and epidermis. in order to improve signal intensity and reduce non-specific background for a subset of mabs, we evaluated the effects of fixation and other steps during sample processing. we found that fixative choice, treatments to remove mucus and bleach pigment, as well as methods for tissue permeabilization and antigen retrieval profoundly influenced labeling by individual antibodies. these experiments led to the development of a step-by-step workflow for determining optimal specimen preparation for labeling whole planarians as well as unbleached histological sections.  we generated a collection of monoclonal antibodies recognizing the planarian intestine and other tissues; these antibodies will facilitate studies of planarian tissue morphogenesis. we also developed a protocol for optimizing specimen processing that will accelerate future efforts to generate planarian-specific antibodies, and to extend functional genetic studies of regeneration to post-transcriptional aspects of gene expression, such as protein localization or modification. our efforts demonstrate the importance of systematically testing multiple approaches to species-specific idiosyncracies, such as mucus removal and pigment bleaching, and may serve as a template for the development of immunological resources in other emerging model organisms. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords planarianregenerationintestinemonoclonal antibody screenimmunohistochemistryimmunofluorescenceissue-copyright-statement© the author 2014 