bluuebunny/crossref_metadata_2025 huggingface dataset:
Double immunocytochemical staining in the study of antibody-producing cells in vivo. Simultaneous detection of cells producing monospecific antibodies and cells producing cross-reacting antibodies in the spleen of rabbits after injection of two related antigens.
Authors: Van Rooijen N , Kors N , Van Nieuwmegen R
Published: April 2011

Rabbits were injected simultaneously with both human gamma globulin (HGG) and bovine gamma globulin (BGG). Sections of spleen tissue were prepared from spleen biopsies taken during the primary or secondary immune response, and incubated simultaneously with horseradish peroxidase (HRP)-HGG conjugate and alkaline phosphatase (AP)-BGG conjugate in order to detect cells containing specific antibodies against one or both of the antigens. After both HRP and AP cytochemistry, cells with a red-stained cytoplasm, cells with a blue-stained cytoplasm, and cells with a violet-stained cytoplasm were detected in the spleen. The red-stained cells had bound the HRP-HGG conjugate, indicating that these cells contained anti-HGG antibodies. The blue-stained cells had bound the AP-BGG conjugate, indicating that these cells contained anti-BGG antibodies. The violet-stained cells had obviously bound both the HRP-HGG conjugate and the AP-BGG conjugate, indicating that these cells contained antibodies cross-reacting with both antigens. Results are compared with earlier studies on the antigenic similarities and differences between HGG and BGG when used as antigens in rabbits.
Unlabeled antibody methods in electron microscopy: a comparison of single and multistep procedures using colloidal gold.
Authors: Gosselin E J , Sorenson G D , Dennett J C , Cate C C
Published: April 2011

A comparative study of five unlabeled antibody methods was conducted on the electron microscopic level using bridging techniques and colloidal gold. The study was based on the principles of the single-step colloidal gold (GLAD) method (Larsson L: Nature 282:743, 1979) and the multistep single- and double-bridge techniques used in postembedding immunoperoxidase procedures (PAP) (Sternberger LA: Immunocytochemistry, 2nd ed. Wiley, New York, 1979). Using medullary thyroid carcinoma and the same lot of primary antiserum (goat anti-calcitonin) for each procedure, it was shown that adequate localization of calcitonin with the single-step GLAD method was attainable only at dilutions of 1:100 or lower. The single-bridge technique using goat anti-calcitonin, sheep anti-goat immunoglobulin (Ig)G, and goat anti-calcitonin and antigen-coated gold, respectively, worked well at dilutions of up to 1:5000 but not at dilutions of 1:10,000, while single- and double-bridging techniques utilizing goat anti-calcitonin, sheep (Sh) anti-goat IgG, and sheep anti-goat IgG-coated gold produced good localization at a 1:10,000 dilution of primary antiserum. A two-step method using goat anti-calcitonin and sheep anti-goat IgG-coated gold, respectively, appeared to be the most sensitive technique, with adequate antigen localization occurring at a dilution of 1:25,000. While in our hands the two-step method appeared superior in sensitivity to the single-bridge IgG-coated gold technique, each method has its own advantages depending on the individual needs of the researcher.
Radioimmunocytochemical localization of retinal S-antigen with monoclonal antibodies.
Authors: Das N D , Ulshafer R J , Zam Z S , Leverenz V R , Shichi H
Published: April 2011

Two monoclonal antibodies (RSA1/83 and RSA2/83) were developed against a homogeneous preparation of bovine retinal S-antigen. The two hybridomas produced by mouse X mouse hybrid myeloma cells secrete immunoglobulin G. Indirect autoradiography on glutaraldehyde-fixed preparations of bovine explants was used to locate the antigenic site. Antibody RSA1/83 recognizes the antigen primarily in the apical region of the rod outer segment, while antibody RSA2/83 located the antigen both in the outer and inner segments of the rod photoreceptor cells. A distinct band of silver grains also appeared along the inner limiting membrane with both antibodies. Control explants showed no specific labeling pattern over the various retinal compartments.
Differences in basement membrane-associated microdomains of type I and type II pneumocytes in the rat and rabbit lung.
Authors: Sannes P L
Published: April 2011

The basement membrane-associated microdomains of type I pneumocytes in rat and rabbit pulmonary alveoli were found to be uniquely different from those of type II pneumocytes in the specific distribution of cytochemically detectable sulfate esters as demonstrated with the high iron diamine (HID) technique at the electron microscopic level. Aldehyde-fixed frozen or Vibratome sections of neonatal and adult lungs were treated with a mixture of the meta and para isomers of N,N-dimethyl-phenylenediamine-HCl in the presence of ferric chloride, which at low pH (1.0) has been previously shown to be highly specific for sulfate esters of glycosaminoglycans and glycoproteins. Reaction product was subsequently enhanced with a thiocarbohydrazide-silver proteinate, postembedding sequence for electron microscopy. Samples of lung parenchyma treated in this fashion were observed to have discrete, electron-dense silver grains associated with the various microanatomical components of pulmonary basement membranes. In the region of the alveolar basement membrane, the lamina rara externa associated with type I cells was observed to contain an abundance of regularly disposed, cytochemically detectable sulfate esters, while the lamina densa and lamina rara interna were diffusely and sparsely reactive by comparison. Quantitatively, 62% of all reactive sites found in the basement membrane region of type I cells were localized in the lamina rara externa. By contrast, the lamina rara externa of type II cells had less than half as many reactive foci indicative of sulfate esters as the same region of type I cell basement membranes. HID-reactive sulfate esters were found evenly distributed within the laminae associated with the basement membrane of type II cells. This cytochemically detectable difference in the sulfate ester composition of basement membrane-associated sulfate ester composition of basement membrane-associated microdomains of type I compared with that of type II pneumocytes may be highly significant when considering known patterns of epithelial renewal in pulmonary alveoli. Since type II cells are known to divide and either remain type II cells or differentiate into type I cells, regional differences in the molecular composition of the alveolar basement membranes and their associated structures may be key determinants of cell-specific processes of cytodifferentiation in the pulmonary alveolus.
Distribution of oncodevelopmental markers in neoplastic cells: therapeutic implications.
Authors: Nakane P K , Watanabe T
Published: April 2011

When the subcellular distribution of secretory component (SC) and carcinoembryonic antigen (CEA) were determined immunoelectronmicroscopically, SC was found on the baso-lateral surface and CEA on the apical surface of the normal gastrointestinal epithelium. In contrast, on the neoplastic cells SC and/or CEA were found all around the cell surface. Taking the change in the distribution of CEA on the neoplastic cells as an advantage, an attempt was made to develop an immunotherapeutic method for adenocarcinoma. The method was based upon an assumption that intravenously injected anti-CEA is not accessible to normal epithelial cells, since the tight junction will act as a barrier for the diffusion of antibodies from the interstitium to the apical cell surface, but the anti-CEA will form immunecomplexes with the CEA on the baso-lateral surface of neoplastic cells. Specifically, CEA-producing human gall bladder carcinoma were transplanted into nude mice. To the tumor-bearing mice, glucose oxidase-labeled anti-CEA was intravenously injected. As a control, glucose oxidase-labeled normal rabbit IgG was injected. This was followed with an injection of NaI. It was found that in those mice injected with the labeled anti-CEA, the size of tumor was reduced as much as 30% within three days. In the controls, the tumor continued to grow. In those injected with the labeled anti-CEA, CEA-anti-CEA immunecomplexes were deposited on the glomerular basement membrane, consequently a search for an insoluble apical antigen is currently made.
Characterization and immunocytochemical application of monoclonal antibodies against enkephalins.
Authors: Cuello A C , Milstein C , Couture R , Wright B , Priestley J V , Jarvis J
Published: April 2011

Monoclonal antibodies were produced following immunization of mice with either [Leu5]enkephalin-bovine serum albumin or [Met5]enkephalin-keyhold limpet hemocyanin conjugates. Two monoclonal antibodies coded NOC1 and NOC2, respectively, were derived. These monoclonal antibodies did not discriminate between Leu- and Met-enkephalin in either radioimmunoassay or immunocytochemistry. NOC1 was characterized in detail. In radioimmunoassay NOC1 displayed about 40% crossreactivity with C-terminal extended Met-enkephalin hexapeptides and 7% with the extended heptapeptide (-Arg-Phe-OH), but did not recognize other endogenous peptides. In immunocytochemistry the NOC1 and NOC2 recognized all well-established "enkephalin immunoreactive sites," but they did not bind to areas known to contain beta-endorphin or high levels of pro-enkephalin. NOC1 was shown to be a suitable tool to demonstrate enkephalin immunoreactive sites by radioimmunocytochemistry utilizing both internally and externally labeled monoclonal antibodies.
Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures.
Authors: Lubit B W
Published: April 2011

Previous immunocytochemical studies in which an antibody specific for mammalian cytoplasmic actin was used showed that a high concentration of cytoplasmic actin exists at neuromuscular junctions of rat muscle fibers such that the distribution of actin corresponded exactly to that of the acetylcholine receptors. Although clusters of acetylcholine receptors also are present in noninnervated rat and chick muscle cells grown in vitro, neither the mechanism for the formation and maintenance of these clusters nor the relationship of these clusters to the high density of acetylcholine receptors at the neuromuscular junction in vivo are known. In the present study, a relationship between beta-cytoplasmic actin and acetylcholine receptors in vitro has been demonstrated immunocytochemically using an antibody specific for the beta-form of cytoplasmic actin. Networks of cytoplasmic actin-containing filaments were found in discrete regions of the myotube membrane that also contained high concentrations of acetylcholine receptors; such high concentrations of acetylcholine receptors have been described in regions of membrane-substrate contact. Moreover, when primary rat myotubes were exposed to human myasthenic serum, gross morphological changes, accompanied by an apparent rearrangement of the cytoplasmic actin-containing cytoskeleton, were produced. Although whether the distribution of cytoplasmic actin-containing structures was influenced by the organization of acetylcholine receptor or vice versa cannot be determined from these studies, these findings suggest that in primary rat muscle cells grown in vitro, acetylcholine receptors and beta-cytoplasmic actin-containing structures may be somehow connected.
Immunohistochemical localization of the immunodominant differentiation antigen lacto-N-fucopentaose III in normal adult and fetal tissues.
Authors: Combs S G , Marder R J , Minna J D , Mulshine J L , Polovina M R , Rosen S T
Published: April 2011

Monoclonal antibodies were produced by immunizing rats with human small cell lung carcinoma (SCLC) cell lines. Monoclonal antibodies 600D11 and 624A12 were found to be directed against the ceramide pentasaccharide that contains the lacto-N-fucopentaose III (LNFP III) sequence of sugars, an isomer of the Lewis A blood group antigen. LNFP III is an immunodominant antigen whose reactivity is maintained in formalin-fixed paraffin-embedded sections (PS). LNFP III has been recognized in a number of human tumors including: SCLC; adenocarcinomas of the breast, gastrointestinal tract, genitourinary tract, and lung; renal cell carcinoma; neuroblastoma; and myelogenous leukemia. We now report the normal adult and fetal tissue distribution of the LNFP III antigen by immunoperoxidase staining on PS utilizing 600D11 and 624A12. Binding was demonstrated in bronchial epithelium and bronchial glands; squamous epithelium of the esophagus; gastric crypts, duodenal enterocytes and Brunners glands; argentaffin cells; jejunal and colonic goblet cells; pancreatic acinar cells; salivary glands; endocervical and exocervical cells; skin epidermis; myelinated motor fibers; cells of the adrenal medulla and anterior pituitary gland; polymorphonuclear leukocytes (PMNs); tissue macrophages and renal proximal tubules and loops of Henle. Staining was localized to cell membranes and within the cytoplasm, with greatest intensity at the apical and basal portions of the cells. These staining patterns were noted in adult and neonatal tissues, and initial expression could be traced to approximately the second trimester of fetal development. Knowledge of the normal tissue distribution of this immunodominant antigenic determinant may offer insight into its structural and functional role in benign and malignant tissues.
Potassium ferricyanide: a stabilizer for horseradish peroxidase.
Authors: Albers F J , Klooster J , van der Want J J
Published: April 2011

Sodium nitroprusside has been used as a stabilizing agent for HRP histochemistry since 1919. However, it is known to have toxic effects orally, intravenously, and subcutaneously. In order to avoid the use of sodium nitroprusside as a stabilizer in HRP histochemistry, we have tested other chemically related compounds to stabilize the reaction product equally well. We will show that potassium ferricyanide is an excellent stabilizer of the chromogen reaction product. In addition, the reaction product remains stable without noticeable changes over a period of several months. As it is far less toxic than sodium nitroprusside, it should be the stabilizer of choice, especially in those laboratories where the histochemical HRP reaction is used frequently.
Histochemical localization of NADP-dependent dehydrogenase activity with four different tetrazolium salts.
Authors: Van Noorden C J , Butcher R G
Published: April 2011

The properties of the four most commonly used tetrazolium salts, neotetrazolium, nitro blue tetrazolium (nitro-BT), tetranitro-BT, and 2-(2-benzothiazolyl-3-(4-phthalhydrazidyl)-5-styryl-te trazolium (BPST), have been compared for their effects on the localization of nicotinamide adenine dinucleotide phosphate (NADP)-dependent dehydrogenases under optimal incubation conditions in cryostat sections of rat liver. Glucose-6-phosphate dehydrogenase has been selected as an example of these dehydrogenases. It was found that the use of nitro-BT and tetranitro-BT, unlike neotetrazolium and BPST, in combination with an exogeneous electron carrier and azide, resulted in localization patterns in agreement with the sites of activity as determined by microchemical techniques. In the absence of an intermediate carrier the localization was very similar to that of NADPH cytochrome c (P450) reductase as demonstrated immunocytochemically. BPST did not properly localize dehydrogenase activity, most probably because of the redistribution of formazan, due to its lack of firm substantivity. Neotetrazolium reduction in nitrogen gave the localization pattern, both in the presence and absence of carrier, of the reductase, suggesting that the transfer of reducing equivalents from the exogenous electron carrier to neotetrazolium proceeds via cellular electron transport systems. The reduction of nitro-BT and tetranitro-BT via intermediate carriers was oxygen sensitive in parenchymal cells, but not in the non-parenchymal liver cells. This oxygen sensitivity could be blocked by azide. With neotetrazolium, oxygen inhibited both carrier-mediated and carrier-independent reactions, effects that were not reversed with azide. Possible mechanisms of action between oxygen, reduced carriers, and tetrazolium salts are discussed.
Quantitative immunocytochemistry of hypothalamic and pituitary hormones: validation of an automated, computerized image analysis system.
Authors: Gross D S , Rothfeld J M
Published: April 2011

A limiting factor in the use of immunocytochemistry in experimental endocrine studies has been the lack of a suitable procedure for quantification of immunoreactive hormones. The objective of the present study was the development of an automated, computerized image analysis system adapted to the quantitative analysis of light microscopic immunocytochemical reaction product. Reaction conditions that result in optimum, standardized, and quantitatively linear development of reaction deposit are described for H2O2 and diaminobenzidine concentrations, antiserum dilutions, and substrate incubation times. In addition, evaluation techniques, including the use of a standard control section to monitor variance and incorporate it into the statistical analysis of the results are documented. For each of the reaction variables, the immunostaining was linear over the range of specific staining. When the optimum conditions were exceeded, marked over-estimations of hormone levels occurred due to the detection of nonspecific background features reaching the detection threshold. Application of this quantitative immunocytochemical (QICC) method to the analysis of variations in hypothalamic and pituitary hormone levels was validated by comparing values obtained with QICC to those with radioimmunoassay (RIA). The relative changes in both hypothalamic gonadotropin-releasing hormone and pituitary luteinizing hormone induced by manipulation of gonadal steroid levels, as measured by RIA and QICC, were highly correlated. Two-way analysis of variance revealed that the two techniques were not significantly different in their detection of changes in either hormone. Thus, under optimally defined conditions, quantitative immunocytochemistry using computerized image analysis has been validated for the accurate measurement of pituitary and brain hormones in precise regions.
A double fluorescence method for determining doxorubicin distribution and vascular supply in the mouse kidney.
Authors: Paschoud N , Bignell C , Reinhold H S
Published: April 2011

Freeze-sectioning followed by freeze-drying completely preserves the topographic distribution of soluble substances, with the additional advantage of permitting permanent mounting. This rapid and effective technique was applied to study the distribution of fluorescence of the cytotoxic drug Doxorubicin in microscopic slices from in vivo treated animals. A fluorescent conjugate of different fluorescence, Blankophor-G-Dextran, which remained in the vessels, was used in the same living animals to characterize the vascular supply in the kidney of treated mice in relation to Doxorubicin distribution.
Synaptic interactions of luteinizing hormone-releasing hormone (LHRH) neurons in the guinea pig preoptic area.
Authors: Silverman A J , Witkin J W
Published: April 2011

Previous studies from many laboratories have failed to demonstrate a significant synaptic input to luteinizing hormone-releasing hormone (LHRH) neurons in the rodent or primate hypothalamus/preoptic area. Having now developed immunocytochemical procedures that result in excellent ultrastructural preservation as well as in retention of antigenicity (Silverman AJ: J Comp Neurol 227:452, 1984), we have reinvestigated the question of the organization of the synaptic arrangements of LHRH neurons in the medial preoptic area of the guinea pig. Afferent inputs to these LHRH neurons include several varieties of axo-somatic and axo-dendritic synapses. Presynaptic terminals contain either round clear vesicles or a mixture of round and flattened vesicles. Most of these terminals, especially when serial sections are examined, contain dense-core granules. Well-defined synaptic clefts are evident and postsynaptic densities can be identified for asymmetrical connections. However, the presence of reaction product in the postsynaptic structure makes it difficult to categorize symmetrical terminals. In addition to these classical inputs, LHRH neurons also enter into complex heterodox synaptic relationships with their neighbors, including somato-dendritic and dendro-dendritic synapses in which the LHRH neuron can be either the pre- or postsynaptic element. These results suggest that complex synaptic relationships might account for the multiple levels of regulation of neurohormone release.
Immunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (shiverer) adult mice with comparisons among fixation methods.
Authors: Cammer W , Sacchi R , Sapirstein V
Published: April 2011

The peroxidase-antiperoxidase technique was used for immunocytochemical localization of carbonic anhydrase in the mouse spinal cord to detect whether this antigen was normally present in myelinated fibers, in oligodendrocytes in both white and gray matter, and in astrocytes, and to determine where the carbonic anhydrase might be localized in the spinal cords of dysmyelinating mutant (shiverer) mice. The most favorable methods for treating tissue were: 1) immersion in formalin-ethanol-acetic acid followed by paraffin embedding, or 2) light fixation with paraformaldehyde and preparation of vibratome sections. Carnoy's solution, followed by paraffin embedding, extracted myelin from the tissue, while aqueous aldehydes, when used before paraffin embedding, reduced staining everywhere except at sites of compact myelin. The latter conclusion was based, in part, on the almost complete loss of this antigen from the shiverer cord, where compact myelin is known to be virtually absent but where membrane-bound carbonic anhydrase was demonstrated enzymatically. When the optimal methods were used with normal mouse cords, carbonic anhydrase was found throughout the white matter columns and in the oligodendrocytes in gray and white matter. The staining of the white matter was attributed to myelinated fibers because of the similarity in distribution to both a histological myelin stain and the immunocytochemical staining for myelin basic protein. In the mutant mice the oligodendrocyte cell bodies and processes, which were stained in all areas of the spinal cord, were particularly numerous at the periphery of the sections. In contrast to the oligodendrocytes, the fibrous astrocytes appeared to lack carbonic anhydrase, or to have lower than detectable levels, since the astrocyte marker, glial fibrillary acidic protein, had a very different distribution from that of carbonic anhydrase. Even finer localization was obtained in vibratome sections, where the antibody against carbonic anhydrase permitted visualization of the processes connecting oligodendrocytes to myelinated fibers in the normal adult spinal cord.
Detection of plasma membrane cholesterol by filipin during microvillogenesis and ciliogenesis in quail oviduct.
Authors: Chailley B , Boisvieux-Ulrich E
Published: April 2011

Using filipin as a probe for the presence of membrane cholesterol, the evolution of cholesterol distribution in the apical plasma membrane was studied during estrogen-induced ciliogenesis in quail oviduct and compared with the distribution of intramembrane particles (IMPs). Ciliary growth is preceded by the first step of microvillus differentiation. Microvilli emerge in membrane domains rich in IMPs and devoid of filipin-cholesterol (f-c) complexes. However growing microvillus membrane shows f-c complexes. During ciliary growth, microvilli lengthen from 0.5 to 2 microns, indicating that the microvillar membrane is not a membrane reservoir for ciliogenesis. During ciliary growth, the characteristic ciliary necklace IMP rows appear progressively at the base of cilia. The first IMP row is organized in a membrane circlet lacking of f-c complexes, whereas the new shaft membrane in the middle of the circlet exhibits numerous complexes. These two different domains of the cilia keep their specificity during ciliary growth. Only the ciliary tip shows fewer complexes than the shaft membrane. The apical membrane of differentiated ciliated cells is thus composed of various domains, the ciliary shaft full of f-c complexes and poor in IMPs, the ciliary necklace is devoid of f-c complexes and rich in IMPs, the microvilli membrane is rich in both IMPs and f-c complexes, and the interciliary membrane is poor in both f-c complexes and IMPs, whereas the undifferentiated cells exhibit an apical membrane in which f-c complexes and IMPs are distributed homogeneously.
Ultrastructural visualization of carbohydrates in oxytalan fibers in monkey periodontal ligaments.
Authors: Takagi M , Yagasaki H , Baba T , Baba H
Published: April 2011

Fullmer's oxytalan fibers appear to be special connective tissue fibers belonging to elastic system fibers. We have ultrastructurally examined carbohydrates in oxytalan fibers in monkey periodontal ligaments after glutaraldehyde fixation and ethylenediaminetetraacetic acid (EDTA) decalcification using: Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for thin-section staining of vicinal glycol-containing complex carbohydrates, and the concanavalin A-ferritin (Con A-ferritin) and Con A-horseradish peroxidase (Con-A-HRP) en bloc staining methods specific for alpha-D-mannosyl and alpha-D-glucosyl groups. PA-TCH-SP stained collagen fibrils weakly to moderately and stained oxytalan fibers moderately. Con A-ferritin and Con A-HRP stained collagen fibrils weakly or moderately and stained oxytalan fibers intensely within the superficial region of specimen blocks. The penetration of staining reagents was improved by prior saponin treatment and/or chondroitinase ABC digestion. Thus, these studies demonstrate that PA-TCH-SP and Con A staining of carbohydrates is very useful in identifying oxytalan fibers at the ultrastructural level and that more carbohydrate components are present in oxytalan fibers than in collagen fibrils.
Characterization of an immunofluorescence technique for the demonstration of coexisting neurotransmitters within nerve fibers and terminals.
Authors: Wessendorf M W , Elde R P
Published: April 2011

Neurotransmitters have been shown to coexist in cell bodies, but demonstrating their coexistence within nerve fibers and terminals has been more difficult. However, two recent reports outlined a simple light-microscopic method by which two neurotransmitters can be shown to coexist in fibers and terminals. The method was identical to that used for immunohistochemical localization of one antigen, except that two primary--secondary antibody systems labeled with two different fluorochromes were used simultaneously. In the present study, a method for the simultaneous visualization of serotonin and substance P was characterized. This method employed an antiserum to serotonin generated in goat in combination with a rabbit-generated antiserum to substance P. These antisera were visualized with secondary antisera raised in swine and conjugated with rhodamine and fluorescein respectively. Spinal cord sections stained by this protocol showed large numbers of fibers fluorescing both red and green. Many of them were in the ventral horn, fewer were around the central canal, and virtually none were in the dorsal horn. The apparent double labeling could be shown not to be the result of cross-reactivity among the antisera, of any inappropriate affinity among the antisera, of green fluorescence by rhodamine, or of red fluorescence by fluorescein. It is concluded that the method provides a simple technique for visualizing fibers and terminals in which serotonin and substance P coexist.
Fiber number and type composition in extensor digitorum longus, soleus, and diaphragm muscles with aging in Fisher 344 rats.
Authors: Eddinger T J , Moss R L , Cassens R G
Published: April 2011

Histochemical (M-ATPase) fiber typing was done on extensor digitorum longus, (EDL), soleus (SOL), and diaphragm (DIA) muscles of barrier-reared Fisher 344 rats obtained at four different ages (3, 9, 28, and 30 months) from the colonies of the National Institute of Aging. In the EDL there are no differences in the percent of type I fibers among the four age groups. The percent of type IIa and IIb fibers also showed no difference between the 3 and 30 month age groups. There was no apparent trend for an increase or decrease in the percent of type IIa or IIb fibers between the four age groups. In both the SOL and DIA muscles the percent of type I fibers was greater in the aged than in the young groups. The percent of type IIa fibers was lower in the 30 month group than in the younger groups for both muscles. The percent of type IIb (DIA) and IIc (SOL) fibers did not change between groups. Total fiber number per cross section of muscle showed no change in the EDL over this age range or in the SOL after 9 months of age. These findings bring into question published results that imply that decreasing fiber number and preferential loss of type II (a and b) fibers are typical aging phenomena.
Localization of Met-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in the gastrointestinal tract of rat and pig.
Authors: Nihei K , Iwanaga T
Published: April 2011

One of the opioid precursor molecules, pre-pro-enkephalin A, contains within it, in addition to Leu-enkephalin (Leu-Enk) and Met-enkephalin (Met-Enk), Met-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-8), which is specific to this precursor. This study deals with the localization of Met-Enk-8-like immunoreactivity in the gastrointestinal tract of rat and pig. Immunoreactivity was identified in intramural nerve elements of rat and pig, and in gut endocrine cells of pig. Immunoreactive (IR) nerve fibers were seen mainly in the myenteric plexus of rat and in both the myenteric and submucosal plexuses of pig. Some IR fibers were dispersed throughout the lamina propria mucosae of rat. Porcine IR endocrine cells were dispersed in the epithelium from the pyloric antrum to the ileum, existing concomitantly with enterochromaffin (EC) cells. Specificity tests revealed that immunoreactivity to Met-Enk-8 antiserum was not influenced by preincubation of the antiserum with Leu-Enk and Met-Enk. This suggests the possibility that pre-pro-enkephalin A is contained in the gastroenteric nerves of rat and pig and in a population of porcine EC cells.
Localization of glucagon-like peptide (GLP) immunoreactants in human gut and pancreas using light and electron microscopic immunocytochemistry.
Authors: Varndell I M , Bishop A E , Sikri K L , Uttenthal L O , Bloom S R , Polak J M
Published: April 2011

The distribution of peptide immunoreactivities predicted from the sequence of the human preproglucagon gene in enteroglucagon (EG; glicentin-like immunoreactant-containing) cells of the human gut and A cells of the pancreas has been determined by light and electron microscopic immunocytochemistry. At light microscopy the application of peroxidase-antiperoxidase and immunogold-silver staining methods has revealed that glucagon-like peptide (GLP-1 and GLP-2) immunoreactivities coexist with a glicentin-related immunodeterminant in human colorectal EG cells and pancreatic A cells. Using single and double colloidal gold probe electron immunocytochemistry, we have been able to show the coexistence of glicentin, GLP-1, and GLP-2 immunoreactivities within single EG cell secretory granules. No morphologic segregation of the proglucagon immunoreactants was observed in EG cells of the colonic mucosa. In pancreatic A cells we have localized GLP-1, GLP-2, and glucagon-[16-29] immunoreactivities solely to the electron-dense core of the secretory granules, whereas glicentin-related immunoreactivity was restricted to the electron-lucent halo. The results obtained in the present study have shown that the peptide immunoreactivities predicted from cDNA sequencing of the human preproglucagon gene are indeed expressed in colorectal EG and pancreatic A cells. The topographical segregation of immunoreactivities in the A cell secretory granule shows that antigenic determinants derived from the C-terminal portion of proglucagon are stored with glucagon in the core of the secretory granule.
Human cytotrophoblast populations studied by monoclonal antibodies using single and double biotin-avidin-peroxidase immunocytochemistry.
Authors: Butterworth B H , Khong T Y , Loke Y W , Robertson W B
Published: April 2011

Single and double biotin-avidin-peroxidase immunocytochemical methods in conjunction with an anti-trophoblast monoclonal antibody 18B/A5 and an anti-HLA-A,B,C monoclonal antibody W6/32 were used to study various human trophoblast populations. Several combinations of peroxidase substrates were tried in the double-labeling procedure. It was concluded that the use of 4-chloro-1 naphthol to develop the primary sequence peroxidase and of 3-amino-9-ethyl carbazole for the second sequence peroxidase was the most suitable. The significant findings were: Monoclonal antibody 18B/A5 proved to be a useful marker for villous as well as nonvillous trophoblast, which facilitated the identification of these cells particularly in the placental bed. The expression of MHC Class I antigens was not confined to extravillous trophoblast but these antigens were also demonstrable on the villous cytotrophoblast proliferating to form new primary villi. Double labeling revealed that many of these cells, particularly those furthest away from the mesenchymal core, expressed both trophoblast and HLA antigens as shown by a mixing of the colors produced by the two reaction products. A large number of these HLA-A,B,C, positive trophoblast cells were found to infiltrate deep into the uterine myometrium. The hypothesis was put forward that these fetal cells could be the ones that are responsible for maternal sensitization.
Histochemical localization of galactose-containing glycoconjugates in sensory neurons and their processes in the central and peripheral nervous system of the rat.
Authors: Streit W J , Schulte B A , Balentine D J , Spicer S S
Published: April 2011

We studied the distribution of sugar residues in the oligosaccharide chains of complex carbohydrates in tissue sections of rat spinal cord, brainstem, and sensory ganglia using twelve lectin-horseradish peroxidase conjugates. Glycoconjugates containing terminal galactose residues were localized apparently in the Golgi apparatus in a population of predominantly small B-type neurons in spinal and trigeminal ganglia. Large A-type neurons rarely showed reactivity with galactose-binding lectins. A cells stained for glycoconjugates with N-glycosidically linked oligosaccharides and glycogen. The central and peripheral processes of the small neurons, mostly unmyelinated C fibers in sensory roots and spinal nerves, contained an abundance of glycoconjugates with terminal alpha-galactose residues. The central projections and terminals of small to medium-sized primary sensory neurons in the spinal and trigeminal ganglia were visualized in Lissauer's tract and the substantia gelatinosa in the spinal cord, and in the spinal trigeminal tract and the nucleus trigeminus in the lower medulla with lectins specific for terminal alpha-galactose residues. In addition, fibers of the solitary system and the area postrema were reactive with these lectins. The peripheral and central nervous system elements with affinity for galactopyranosyl-specific lectins correspond in distribution with neuroanatomical regions thought to be involved in the transmission and relay of somatic and visceral afferent inputs such as pain and temperature. Such specific localization of a glycosubstance to a distinct subpopulation of neurons and their peripheral and central processes suggests that the particular glycoconjugate may be of physiological significance.
Quantitative histochemical determination of muscle enzymes: biochemical verification.
Authors: Martin T P , Vailas A C , Durivage J B , Edgerton V R , Castleman K R
Published: April 2011

We established quantitative histochemical assays for the enzymatic activity of succinate dehydrogenase and alpha-glycerol phosphate dehydrogenase for cat skeletal muscle. A computer-enhanced image analysis system was used to quantitate the histochemical enzyme-activity reaction products. We describe a series of experiments that verify the reliability and validity of the assays. Histochemically determined enzyme activities were linear with respect to tissue thickness and reaction time. Biochemically determined enzyme activities were also linear with respect to tissue thickness and incubation time. Consecutive tissue sections, assayed either histochemically or biochemically, were used to establish a linear regression equation that allowed quantitative histochemically determined reaction rates, measured in optical density per minute, to be calibrated as nanomoles per minute.
Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies.
Authors: Mullink H , Henzen-Logmans S C , Tadema T M , Mol J J , Meijer C J
Published: April 2011

A number of fixation and decalcification procedures were evaluated to determine their suitability for immunohistochemistry on trephine samples of bone marrow after paraffin embedding. In particular, the immunoreactivity of antigens characteristic for various hematopoietic cell lines (immunoglobulin heavy and light chains for plasmacytoid cells; elastase for neutrophil myeloid cells; lysozyme, alpha-1-antitrypsin and alpha-1-antichymotrypsin for hystiocytic cells; leukocyte common antigen for lymphocytes; hemoglobin and glycophorin A for erythroid cells; Factor VIII-related antigen for thrombocytoid cells) as well as some antigens specific for epithelial tumors (CEA, 115D8, and keratin) were investigated. Fixation in a mercuric chloride-formaldehyde mixture followed by decalcification in acetic acid-formaldehyde-saline proved to be the best procedure for antigen preservation and retention of morphologic detail. Moreover, there is no need of trypsinization when using this procedure. The only exception was Factor VIII-related antigen in megakaryocytes, which was best demonstrated in trypsin-digested sections of formalin-fixed and acetic acid-decalcified biopsies.
Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis.
Authors: Neutra M R , Ciechanover A , Owen L S , Lodish H F
Published: April 2011

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.
Demonstration of myoglobin and CK-M in myocardium. Comparison of five fixation methods and three immunohistochemical techniques.
Authors: Moran M M , Siegel R J , Said J W , Fishbein M C
Published: April 2011

The results of immunohistochemical staining vary depending on the tissue, fixative, antigen-antibody system, and immunohistochemical staining methods used. The purpose of this study was to evaluate the effect of different methods of fixation, different antigen-antibody systems, and different immunohistochemical methods on immunohistochemical staining of myocardium. Samples of normal fresh canine myocardium from six dogs were fresh frozen and fixed in 10% neutral buffered formalin, Bouin's, Bayley's and Carnoy's fixatives. Immunohistochemical staining for myoglobin and creatine kinase M was performed using the ABC (avidin-biotin complex) and indirect peroxidase-antiperoxidase (PAP) techniques. Tissues fixed in formalin showed the most intense specific staining for both antigens with the least background and nonspecific staining. All other fixation methods and frozen section techniques gave a more variable degree of specific positive staining and substantial background staining and/or nonspecific staining. ABC and PAP techniques gave similar results with both antigen-antibody systems and with each fixation method. Thus, no differences in specificity or sensitivity were observed between ABC and PAP techniques. Differences in staining intensity and pattern were related primarily to differences in fixation methods.
Internalization and delivery to lysosomes of hydrazide horseradish peroxidase, a covalent membrane probe.
Authors: Storrie B , Ferris A L
Published: April 2011

Hydrazide horseradish peroxidase, (hydHRP), a hydrazide derivative of the common cytochemical tracer HRP, was covalently coupled to the surface of periodate-treated Chinese hamster ovary (CHO) cells and used to study the distribution and internalization of plasma membrane glycoconjugates. The Schiff-base coupling of hydHRP to the cell surface at 4 degrees C had little effect on cell viability. After coupling, cells were washed at 4 degrees C and the subcellular distribution of hydHRP was determined immediately or after incubation at 37 degrees C. Within 1 hr, hydHRP was observed to cap over pseudopodal-like extensions and then accumulate over a 2.5 h period in a punctate to perinuclear staining pattern over the cell body. By electron microscopy, the pseudopodal-like regions were found to be areas of extensive cell surface invaginations, rich in microfilaments. HydHRP internalized over a 2.5 to 18 hr period was observed in smooth vesicles resembling pinosomes/endosomes, multivesicular bodies (lysosomes), and small perinuclear vesicles. Little, if any, hydHRP activity was detected in association with elements of Golgi apparatus. By cell fractionation in 10% Percoll gradients, hydHRP was found to have accumulated in prelysosomal endocytic vesicles and lysosomes. For cells that were first surface labeled with 125I at 4 degrees C and then conjugated with hydHRP, little, if any, cotransport of the 125I label with hydHRP was observed. Over the entire capping and internalization period, most hydHRP activity remained membrane associated. Overall, these results indicate that the dominant intracellular transport route for a covalent membrane probe, hydHRP glycoconjugate, is similar if not identical to that previously reported for the solute probe native HRP (16) in CHO cells. HydHRP internalization provides further evidence for the independent sorting of proteins in endocytic transport.
Use of fluorescence flow cytometry to study the binding of various ligands to platelets.
Authors: Dunstan R A
Published: April 2011

Fluorescence flow cytometry can be used to analyze the binding of different ligands to platelets. However careful choice of volume gates is essential in selecting the population of platelets for analysis. The use of fluorescein isothiocyanate conjugated to staphylococcal protein A or F(ab')2 fragments of immunoglobulin G anti-immunoglobulin offers no advantage in sensitivity or specificity in fluorescence studies of platelets and prefixation of washed platelets with paraformaldehyde has no effect on nonspecific fluorescence. The application of this technology to platelets facilitates quantitation of fluorescence intensity and may yield additional useful information.
Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry.
Authors: Nanci A , Bendayan M , Slavkin H C
Published: April 2011

Mouse secretory ameloblasts express a number of enamel proteins, which have been divided into amelogenin and enamelin subfamilies. We have used polyclonal antibodies to murine amelogenins to reveal enamel proteins in mouse ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabeling was detected over the extracellular enamel matrix and over the rough endoplasmic reticulum, the saccules of the Golgi apparatus, and the secretory granules of the ameloblasts. In addition, some lysosome-like granules were also labeled. Only background labeling was obtained over mitochondria, nuclei, cytosol, adjacent odontoblasts, and dentin. Quantitation of the intensity of labeling showed the presence of an increasing gradient along the secretory pathway, which may correspond to the concentration or the maturation of these proteins as they are processed by the cell. These findings indicate that the ameloblast displays an intracellular distribution of its secretory products similar to that of other merocrine secreting cells. The presence of enamel proteins in lysosomes suggests that crinophagy and/or resorption occurs in these cells.
Cytochemical localization of dipeptidyl peptidase II (DPP-II) in mature guinea pig sperm.
Authors: Talbot P , Dicarlantonio G
Published: April 2011

Guinea pig sperm contain the exopeptidase dipeptidyl peptidase II (DPP-II). Our purpose was to localize this enzyme in sperm at the ultrastructural level using lysylalanyl-4-methoxy-beta-naphthylamide which is the specific synthetic substrate for DPP-II. In substrate incubated sperm, reaction product was located within the acrosome. It was concentrated over the light staining area which forms the prominent dorsal bulge of the acrosome. Within this area there are spherical zones of moderate electron density which had little or no reaction product. Control sperm did not have reaction product. We conclude that DPP-II is restricted to a compartment within the guinea pig sperm acrosome.