bluuebunny/crossref_metadata_2025 huggingface dataset:
The osmium tetroxide-p-phenylenediamine procedure reveals the chromatid cores and kinetochores of meiotic chromosomes by light and electron microscopy.
Authors: Antonio C , González-García J M , Page J , Suja J A , Stockert J C , Rufas J S
Published: April 2011

We analyzed first-metaphase meiotic chromosomes of the grasshopper Chorthippus jucundus by two different methods, i.e., a silver impregnation technique and the osmium tetroxide-p-phenylenediamine (Os-PPD) procedure. The former was applied on squashed testes previously fixed in ethanol-acetic acid, whereas for Os-PPD the material was not subjected to any previous extraction treatment but was fixed in OsO4, treated with PPD, and embedded in Epon 812. Both techniques revealed chromatid cores and kinetochores regardless of the processing of the material (squashed or sectioned). Unstained Os-PPD sections were analyzed by light microscopy and transmission electron microscopy (TEM). The Os-PPD technique provided a high contrast of chromatid cores and kinetochores in relation to the chromatin, which revealed a low electron density. To determine the Os-PPD reaction mechanism, the PAS procedure, as well as scanning electron microscopy (SEM) backscattering and SEM X-ray microanalysis, was performed on sections. By use of the Os-PPD-PAS procedure, glycol groups formed by oxidation of osmium bound to aromatic substrates were detected in chromatid cores and kinetochores by brightfield and fluorescence microscopy. A high Z contrast was detected in these structures by backscattered electron imaging. SEM X-ray microanalysis showed osmium and phosphorus to be the main elements present on the chromatid cores. Taking into account the known reactivity of OsO4 and the present results, the possible participation of nucleic acids as well as proteins in the Os-PPD reaction mechanism and in the composition of chromatid cores and kinetochores is discussed.
Extracellular matrix characterization during healing of full-thickness wounds treated with a collagen/elastin dermal substitute shows improved skin regeneration in pigs.
Authors: Lamme E N , de Vries H J , van Veen H , Gabbiani G , Westerhof W , Middelkoop E
Published: April 2011

We investigated the architecture of the extracellular matrix (ECM) during healing of full-thickness wounds in the pig. Two different treatments, one based on epidermal transplantation (split skin mesh grafts, SP wounds) and one consisting of a combination of epidermal transplantation and a dermal matrix substitute (MA wounds) were compared. The dermal matrix consisted of native bovine collagen coated with elastin hydrolysate. The latter treatment reduced wound contraction and improved tissue regeneration. The expression patterns of fibronectin, von Willebrand factor, laminin, chondroitin sulfate, and elastin, detected by immunohistochemistry, were examined in time and indicated different stages of healing. During the early phase of healing the dermal matrix induced more granulation tissue, a different fibronectin expression pattern, and rapid vascular cell ingrowth (von Willebrand factor). Furthermore, in the MA wounds chondroitin sulfate was detected earlier in the basement membrane and fibronectin staining disappeared more rapidly. During later stages of healing, chondroitin sulfate expression was selective for areas in which ECM remodeling was active; in these specific areas elastin staining reappeared. ECM remodeling and elastin regeneration occurred both in the upper and lower dermis for the MA wounds but only in the upper dermis for the SP wounds. Electron microscopic evaluation of the wounds after 2 weeks showed many myofibroblasts in the SP wounds, whereas in the MA wounds cells associated with the dermal matrix had characteristics of normal fibroblasts. The results suggest that the biodegradable dermal matrix served as a template for dermal tissue regeneration, allowed faster regeneration, and improved the quality of healing in large full-thickness skin defects.
Reconstructed three-dimensional images of flow-sorted nuclei hybridized with chromosome-specific probes.
Authors: Matsuta M , Matsuta M , Hayashi S , Yasumi S , Sasaki K , Kuroda Y , Nishiya I
Published: April 2011

We demonstrated that the three-dimensional (3-D) locational and morphological differences of chromosome 17 are dependent on each cell cycle phase in the clinical materials. Cell suspensions prepared from hypertrophied tonsil were hybridized with chromosome 17 whole painting probe or its centromeric probe and the probes were detected with fluorescein isothiocyanate. Then the cells were sorted from G(0+1), S-, and G(2+M)-phase fractions by flow cytometry and observed by confocal laser scanning microscopy to obtain the serial optical sections. The 3-D images were obtained by assembling these sections using a computerized image analysis device. The distribution of centromeric copies was analyzed statistically, and the data values were not a population of random distribution within a sphere. The copies were observed in the periphery of the nuclei in G(0+1)- and S-phase. The 3-D images revealed that chromosome 17 was oval in shape in the G(0+1)-phase nucleus, and was changing into a flame shape in the S-phase, with arms stretching out along the nuclear membrane, and looked bush shaped in G2-phase. The eccentric distribution of chromosome 17 in G(0+1)- and S-phase nuclei may reflect the optimal efficiency of incorporating and/or releasing essential materials and products.
Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining.
Authors: Hunyady B , Krempels K , Harta G , Mezey E
Published: April 2011

The biotinyl-tyramide substrate of the horseradish peroxidase enzyme has been recently introduced to amplify immunohistochemical signals. We applied either fluorochromeor biotin-conjugated tyramine to improve the detection of different antigens in sections of rat stomach, pancreas, and hypothalamus. A ten- to 100-fold increase in staining efficiency was achieved, depending on the antibody, with either fluorescent or peroxidase detection systems. The amplification method was particularly useful for increasing a weak signal of conventional immunostaining caused by suboptimal tissue fixation. At a very low concentration of the primary antibody, the antigen can no longer be detected by a conventional fluorescent secondary antibody but is still detectable after amplification. When an antibody is used at this very low concentration and is detected by a fluorescent amplification method, another primary antibody, raised in the same host species, can be used and demonstrated with a different fluorochrome in subsequent conventional immunostaining of the same section. In this way it becomes possible to immunostain the same section with two different primary antibodies raised in the same host species. Samples for such double immunostaining are demonstrated here using pairs of monoclonal antibodies (to tyrosine hydroxylase and oxytocin) in the hypothalamus and polyclonal antibodies (to glucagon and neurofilament M) in sections of rat pancreas. Because in many cases the availability of antibodies is limited, the amplification method can be a quick and efficient tool for double immunostaining with antibodies from the same host species.
Upregulation of dynamin II expression during the acquisition of a mature pancreatic acinar cell phenotype.
Authors: Cook T A , Mesa K J , Gebelein B A , Urrutia R A
Published: April 2011

Members of the dynamin superfamily are GTPases which have been shown to support receptor-mediated endocytosis in vivo and bind to growth factor receptor-associated proteins in vitro. In acinar cells of the pancreas, receptor-mediated endocytosis is very important for the recycling of membranes after secretory granule release. Therefore, characterization of the molecular machinery responsible for this process is critical for a better understanding of this phenomenon. In this study we sought to determine the expression pattern of the endocytic GTPase dynamin II during pancreatic acinar cell differentiation in developing rat embryos and in dexamethasone-treated AR42J cells using Western blot, Northern blot, and immunocytochemical analyses. During pancreatic development, dynamin immunoreactivity is almost undetectable until day E17 but undergoes significant upregulation in acinar cells starting at E18. In addition, the levels of dynamin mRNA and protein in AR42J cells increase approximately threefold during dexamethasone-induced acinar differentiation. The increase in dynamin levels that occurs in both embryonic pancreatic cells and dexamethasone-treated AR42J cells correlates with the establishment of a more differentiated acinar phenotype. Therefore, these results suggest a potential role for dynamin in supporting receptor-mediated endocytosis in mature pancreatic acinar cells.
Microfibril-associated glycoprotein-1 (MAGP-1) is specifically located on the beads of the beaded-filament structure for fibrillin-containing microfibrils as visualized by the rotary shadowing technique.
Authors: Henderson M , Polewski R , Fanning J C , Gibson M A
Published: April 2011

This study used immunoelectron microscopic techniques to define the ultrastructural location of MAGP-1 on the fibrillin-containing microfibrils of the ocular zonule. A specific anti-MAGP-1 monoclonal antibody (MAb), 11B, was produced that did not crossreact with fibrillin-1 or other microfibrillar proteins. MAb 11B was shown by immunofluorescence to localize intensely to zonular tissue. Postembedding immunoelectron microscopy showed that MAGP-1 was associated with microfibrils throughout the zonule, with the exception of a narrow band of microfibrils at the junction with the lens capsule. With preembedding labeling, the anti-MAGP-1 MAb was found to localize in a crossbanding pattern, at intervals of about 50 nm, to microfibrils throughout the zonule and along bundles of microfibrils in surrounding vitreous tissue. Rotary shadowing of isolated microfibrils showed a "beads on a string" morphology with a periodicity of about 50 nm. With immunogold labeling, the anti-MAGP-1 antibody specifically localized on the beads in a symmetrical manner. Occasionally two gold partides were attached to the same bead, suggesting that multiple MAGP-1 molecules were present in the structure. The results indicate that MAGP-1 is intimately and regularly associated with the bead regions of fibrillin-containing microfibrils. The findings are consistent with a major structural role for MAGP-1 in microfibril biology.
Histochemical and immunohistochemical detection of neurons that produce nitric oxide: effect of different fixative parameters and immunoreactivity against non-neuronal NOS antisera.
Authors: Gonzalez-Hernandez T , Perez de la Cruz M A , Mantolan-Sarmiento B
Published: April 2011

This study focused on two points concerning the histochemical and immunohistochemical detection of neurons that produce nitric oxide (NO): (a) the effect of fixation and other methodological parameters on the staining pattern of both NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, and (b) the possibility that neurons display immunoreactivity against NOS antisera obtained from non-neuronal sources. Frontal sections of rat brains, fixed with 4% paraformaldehyde according to different protocols, were processed for single and double labeling using NADPH-d histochemistry and neuronal (nNOS), macrophagic (macNOS), and endothelial (eNOS) NOS immunohistochemistry. Our results show that variations in the fixative schedule, even within standard parameters, produce qualitative and quantitative changes in NADPH-d labeling. The effect of fixative on weakly stained neurons is different from that on heavily stained neurons. In subfixed brains, a large number of NOS-positive neurons lose their NADPH-d activity, whereas NOS immunolabeling remains unaltered. This finding may be particularly interesting in morphological studies that compare NADPH-d activity under experimental conditions that can affect brain perfusion. On the other hand, many cortical and subcortical neurons show macNOS immunoreactivity, most of it colocalized with nNOS.
Basement membrane abnormalities in human eyes with diabetic retinopathy.
Authors: Ljubimov A V , Burgeson R E , Butkowski R J , Couchman J R , Zardi L , Ninomiya Y , Sado Y , Huang Z S , Nesburn A B , Kenney M C
Published: April 2011

Vascular and parenchymal basement membranes (BMs) are thickened in diabetes, but alterations in individual BM components in diabetic eyes, especially in diabetic retinopathy (DR), are obscure. To identify abnormalities in the distribution of specific constituents, we analyzed cryostat sections of human eyes obtained at autopsy (seven normal, five diabetic without DR, and 13 diabetic with DR) by immunofluorescence with antibodies to 30 BM and extracellular matrix components. In non-DR eyes, no qualitative changes of ocular BM components were seen. In some DR corneas, epithelial BM was stained discontinuously for laminin-1, entactin/nidogen, and alpha3-alpha4 Type IV collagen, in contrast to non-DR corneas. Major BM alterations were found in DR retinas compared to normals and non-DR diabetics. The inner limiting membrane (retinal BM) of DR eyes had accumulations of fibronectin (including cellular) and Types I, III, IV (alpha1-alpha2), and V collagen. The BM zone of new retinal blood vessels in neovascularized areas accumulated tenascin and Type XII collagen, whereas normal, diabetic, and adjacent DR retinas showed only weak and irregular staining. In preretinal membranes, perlecan, bamacan, and Types VI, VIII, XII, and XIV collagen were newly identified. Diabetic BM thickening appears to involve qualitative alterations of specific BM markers at an advanced disease stage, with the appearance of DR.
Stage-specific expression of RESP18 in the testes.
Authors: Schiller M R , Darlington D N
Published: April 2011

RESP18 (regulated endocrine-specific protein of 18 KD) is an endoplasmic reticulum (ER) protein that was identified by coordinate dopaminergic regulation with pro-opiomelanocortin in the rat neurointermediate pituitary. Many attributes of RESP18 suggest an important function in neuroendocrine cells. Several neuropeptides, growth factors, and enzymes involved in biosynthesis of classical chemical neurotransmitters, have been identified in germ cells, Sertoli cells, and spermatozoa. In this study, screening of reproductive tissues revealed high levels of RESP18 protein and mRNA in the testes but not in ovaries or epididymis. The testes and sperm expressed 18-KD RESP18 and a unique 19-KD isoform. To better understand RESP18 expression in the testes, we have examined the stages of the cycle of the seminiferous epithelium by immunohistochemistry and Western blot analyses. Immunohistochemical analysis showed that RESP18 protein was expressed exclusively in spermatocytes and maturing spermatids. RESP18 protein was expressed at high levels in Step 1-8 round spermatids, in which the PC4 prohormone convertase, nerve growth factor, and proenkephalin are also expressed. Western blots, Northern blots, and indirect immunofluorescence staining demonstrated RESP18 expression in sperm.
Cytochemical localization of ecto-ATPases in rat neurohypophysis.
Authors: Thirion S , Troadec J D , Nicaise G
Published: April 2011

We studied the distribution of Ca(2+)- or Mg(2+)-dependent ATPase activity in rat neurohypophysis using the lead cytochemical method of Ando et al. In electron microscopy, precipitates were found lining the outer surface of the plasma membrane surrounding nerve endings and pituicytes. These precipitates were believed to represent the activity of ecto-ATPases (as opposed to Ca pump ATPases) for the following reasons: there was equal activation by Ca2+ in the absence of Mg2+ or Mg2+ in the absence of Ca2+; the effects of the two ions were not additive; there was activation by ATP or GTP; and there was resistance to glutaraldehyde fixation, to high (10 mM) Ca2+ concentrations, and to various inhibitors such as NEM, vanadate, oligomycin, quercetin, p-chloromercuribenzoate, ouabain, and levamisole. Cytosolic activity observed in certain nerve endings in the same conditions of incubation but more sensitive to NEM is also described and discussed.
Embryonic mouse lung epithelial progenitor cells co-express immunohistochemical markers of diverse mature cell lineages.
Authors: Wuenschell C W , Sunday M E , Singh G , Minoo P , Slavkin H C , Warburton D
Published: April 2011

Developmental expression of marker genes representative of different mature cell types can be used to study differentiation of cell lineages. We used immunohistochemistry to study expression in developing mouse lung of calcitonin gene-related peptide (CGRP), Clara cell 10-KD protein (CC10), and surfactant protein-A (SP-A), markers that are differentially expressed in neuroendocrine cells, Clara cells, and Type II alveolar cells. Two distinct developmental phases were revealed. The earlier phase (embryonic days 13-15; E13-E15) was characterized by CGRP, CC10, and SP-A immunostaining in all epithelial cells of the distal airways, with the three patterns being virtually identical in adjacent sections. The later phase (E16-E18) was characterized by emergence of staining of the differentiated cell types. These expression patterns were recapitulated in serumless organ culture, demonstrating that information necessary to generate both phases of gene expression is present within the lung analage by E11. We conclude that CGRP, CC10, and SP-A are co-expressed in most or all cells of the distal lung epithelium at E13-E15 and later become restricted to different cell lineages. This transient expression in progenitor cells of gene products characteristic of diverse differentiated cell types may reflect an underlying mechanism of gene regulation.
Localization of apoptotic cells by direct immunogold detection of digoxigenin-labeled genomic DNA in semithin sections.
Authors: Van Nassauw L , Schrevens A , Harrisson F , Callebaut M
Published: April 2011

In this study, which correlates apoptosis with avian ovarian physiology, we modified an in situ DNA nick end-labeling method using immunogold reagents to detect apoptotic cells in semithin sections of quail ovaries embedded in glycol methacrylate resin. Special attention was paid to the prevention of background staining. The results are comparable with those of the ApopTag peroxidase kit in paraffin-embedded ovaries.
Concurrent immunohistochemical staining of tumor-infiltrating lymphocytes and carcinoma-associated T (Thomsen-Friedenreich)/Tn antigens in human breast carcinoma.
Authors: Wang B L , Springer G F , Kaufman M W
Published: April 2011

The composition of tumor-infiltrating lymphocytes (TIL) often reflects the host's immune response to the tumor. To study the relationship of TIL and carcinoma-associated T/Tn antigens in breast carcinoma, a straightforward concurrent immunoenzyme staining procedure was developed. Fresh tissue was directly fixed in a zinc-based fixative to preserve lymphocyte markers and then routinely embedded in paraffin. The TIL subtypes in the sections were identified in the first immunostaining cycle by reaction with a monoclonal antibody (MAb) to lymphocyte markers CD3, CD4, CD8, CD19, or CD56, followed by a modified avidin-biotin procedure and diaminobenzidine tetrahydrochloride-H2O2 for color development. This was followed by paraformaldehyde fixation to block antibody crossreactivity. The T and Tn antigens on carcinoma cells were then demonstrated in a second staining cycle by reaction with an MAb against T or Tn antigen, followed by an indirect immunoalkaline phosphatase procedure and corresponding substrate systems for color development. The distinguishable brown color for TIL and blue or red color for T or Tn antigen enabled us to identify the TIL subsets and to describe their relations with T/Tn antigen expression in situ. This approach may contribute to better understanding of the patients' immune defenses against their tumor and aid in prognostication.
Validation of the S-phase specificity of histone (H3) in situ hybridization in normal and malignant cells.
Authors: Gown A M , Jiang J J , Matles H , Skelly M , Goodpaster T , Cass L , Reshatof M , Spaulding D , Coltrera M D
Published: April 2011

Several different methods of measuring proliferation indices have been developed, including measurements of cellular DNA content (flow cytometry), S-phase incorporation of thymidine analogues into DNA (e.g., tritiated thymidine and 5'-bromodeoxyuridine), and immunostaining of cell cycle-restricted proteins (e.g., Ki-67 antigen and PCNA). Theoretical and practical problems with each method have made it difficult to compare absolute proliferation rates among cells of different lineages and degrees of malignancy. More recently, in situ hybridization (ISH) for histone 3 (H3) mRNA has been introduced. We used a double labeling method for comparing H3 mRNA expression and S-phase incorporation of 5'-bromodeoxyuridine (BrdU) to determine if H3 mRNA expression was tightly associated with S-phase in a variety of malignant and nontransformed cell types. In addition, labeling results were compared in methacarn- and formalin-fixed tissues to extend the potential usefulness of H3 ISH, using a postfixation technique for the alcohol-fixed specimens. As expected for a cumulative marker, variation was noted in the percentage of the BrdU-positive cells double labeled with H3 ISH (53-89%), depending on cell type and length of BrdU incubation. In contrast, the percentage of the H3 ISH-positive cell population double labeled for BrdU was independent of the cell type of BrdU incubation time (mean 78%). Similarly, a consistent percentage of H3 ISH-positive cell populations was double labeled for BrdU in normal tissues (mean 97%). These findings support a well-conserved timing mechanism for H3 mRNA expression and DNA replication. We conclude that H3 ISH is an extremely accurate technique for assessment of S-phase cell proliferation indices.
Differential distribution of Met and epidermal growth factor receptor in normal and carcinogen-treated rat liver.
Authors: Huitfeldt H S , Skarpen E , Lindeman B , Becher R , Thrane E V , Schwarze P E
Published: April 2011

Transforming growth factor-alpha (TGF-alpha) and hepatocyte growth factor (HGF) are strong hepatocyte mitogens and important regulators of liver regeneration. The TGF-alpha receptor EGFr appears primarily to mediate a proliferative signal, whereas mitogenic, motogenic, and morphogenic effects have been attributed to activation of the HGF receptor Met. We have studied the localization of Met and EGFr in normal and carcinogen-treated rat livers. Oval cells and preneoplastic lesions were induced by diethylnitrosamine initiation, followed by promotion with 2-acetylaminofluorene combined with a partial hepatectomy. Different liver cell populations and their receptor expression were characterized by two-color immunofluorescence and confocal laser scanning microscopy. Hepatocytes were detected by keratin K8 staining, and oval cells and bile ducts were recognized by keratin K19 expression. Enzyme-altered preneoplastic lesions ere identified by expression of placental glutathione S-transferase (GST-pi). Staining for these cellular markers was combined with immunodetection of EGFr and Met. Normal liver exhibited strong staining for EGFr in hepatocytes, whereas blood vessels, bile ducts, and some sinusoidal cells were Met-positive. In carcinogen-treated livers, oval cells showed Met but not EGFr immunostaining. GST-pi-positive foci displayed EGFr immunostaining at a similar intensity as surrounding hepatocytes, whereas Met was not detected. Our data indicate that putative liver cells (oval cells) have a growth receptor phenotype similar to that of bile ducts, whereas preneoplastic live lesions appear hepatocyte-like. These results indicate that the preferential proliferation of preneoplastic liver lesions compared to surrounding hepatocytes is not associated with an altered EGFr or Met phenotype.
Epitope enhancement for immunohistochemical demonstration of tartrate-resistant acid phosphatase.
Authors: Janckila A J , Lear S C , Martin A W , Yam L T
Published: April 2011

We have developed a monoclonal antibody (9C5) for immunohistochemical localization of tartrate-resistant acid phosphatase (TRAcP). This antibody reacts with a denatured epitope of TRAcP and requires enhancement methods to promote antigenicity in paraffin-embedded tissues. We used this antibody to systematically examine proteolytic digestion and heat denaturation conditions for epitope enhancement in both paraffin sections and fixed smears. The goal was to increase the sensitivity of the immunohistochemical stain for TRAcP. Optimal conditions for proteolytic digestion were established. Denaturation in a conventional boiling water bath was compared to microwave irradiation in several commonly used solutions. Immunohistochemistry was compared directly to TRAcP cytochemistry in fixed smears from hairy cell leukemia specimens to gauge the level of sensitivity of our improved method. Attempts were made to "retrieve" the 9C5 epitope from overfixed tissues and aged smears. Maximal immunoreactivity of TRAcP was achieved by microwave irradiation in a citrate or Tris buffer of pH 6.0-8.0 without the need for a subsequent protease digestion step. With this method of epitope enhancement, immunohistochemistry with antibody 9C5 was as sensitive as direct cytochemical staining of TRAcP activity. However, once a tissue specimen had been overfixed or a smear stored for a year or more, the 9C5 epitope was no longer retrievable. The key element in epitope enhancement for 9C5 immunohistochemistry is heat denaturation of the target epitope. Immunohistochemistry of TRAcP in paraffin sections would be a great asset to the study of specialized forms of the monocyte/macrophage lineage and to the process of macrophage activation. It would also provide another means for more precise evaluation of residual disease in bone marrow of patients treated for hairy cell leukemia.
Carbonic anhydrase isoenzyme II is located in corticotrophs of the human pituitary gland.
Authors: Parkkila A K , Parkkila S , Rajaniemi H
Published: April 2011

We studied the location of carbonic anhydrase (CA) isoenzymes I, II, and VI in human pituitary gland using specific antisera in conjunction with immunoblotting, immunoperoxidase, and double immunofluorescence staining techniques. Stainings with anti-CA II serum showed intense cytoplasmic reaction in the anterior lobe of the pituitary gland. Double immunofluorescence staining was used to identify the cells that expressed CA II. Confocal laser scanning microscopy revealed that, of the anterior pituitary hormones studied, ACTH coincides mainly with CA II in these cells. Stainings with anti-CA I and VI sera were negative in the endocrine cells of the pituitary gland. Western blotting of the pituitary gland with anti-CA II revealed a distinct 29-KD polypeptide band corresponding in molecular weight to CA II, suggesting that the antiserum does not detect any nonspecific protein. Anti-CA I serum similarly showed a major 29-KD band, possibly recognizing the enzyme, which is abundantly present in erythrocytes. The results indicate that CA II is expressed in corticotrophs of human pituitary gland, in which its physiological role may be linked to the regulation of optimal pH in the secretory vesicles for the cleavage of ACTH from its precursor.
A simple, reliable, and sensitive method for nonradioactive in situ hybridization: use of microwave heating to improve hybridization efficiency and preserve tissue morphology.
Authors: Lan H Y , Mu W , NG Y Y , Nikolic-Paterson D J , Atkins R C
Published: April 2011

The digestion of fixed tissue sections is a critical step in the optimization of any in situ hybridization protocol. We describe a novel application of microwave oven heating to optimize mRNA detection in paraformaldehyde-fixed tissues by in situ hybridization using digoxigenin-labeled probes. This technique replaces protease digestion of fixed tissue sections with 10 min of microwave pretreatment, followed by either conventional hybridization or hybridization involving microwave incubation. This new technique has several advantages over the standard protease treatment-based methods presently in use. (a) Microwave oven heating is a simple, rapid, and highly reproducible technique. (b) Microwave pretreatment significantly increased the hybridization signal and reduced the background compared to conventional protease digestion. Consequently, the hybridization time required to obtain optimal mRNA detection was reduced to 30 min. (c) Ten minutes of microwave pretreatment produced an optimal hybridization signal in six different tissues using a variety of probes, demonstrating the general applicability of this technique. (d) Microwave heating of the probe during the hybridization step itself further reduced the hybridization time and substantially enhanced the hybridization signal obtained from proteinase K-digested tissue. (e) Microwave pretreatment caused no discernible loss of fine cell structure and tissue morphology compared to untreated tissue sections. In conclusion, microwave oven heating can replace the complicated strategies and poor reproducibility of protease treatment of tissue sections, resulting in a simple, rapid, more reliable and sensitive method that has general applicability for in situ hybridization.
Comparison of LR White and Unicryl as embedding media for light and electron immunomicroscopy of chromaffin cells.
Authors: Goping G , Kuijpers G A , Vinet R , Pollard H B
Published: April 2011

LR White and Unicryl are members of the same family of acrylic embedding resins and are very suitable for "on grid" postembedding immunogold labeling. We studied the ultrastructure of LR White- and Unicryl-embedded cultured chromaffin cells and the immunolocalization of three chromaffin cell proteins, the enzymes dopamine-beta-hydroxylase (DbetaH) and tyrosine hydroxylase (TH), and the membrane fusion and Ca2+ channel protein synexin (annexin VII). We report here that Unicryl is preferable to LR White as an embedding medium for electron microscopy when osmium tetroxide fixation is omitted. The basis for this distinction is better ultrastructural preservation and improved immunodetection efficiency.
Distribution of the large aggregating proteoglycan versican in adult human tissues.
Authors: Bode-Lesniewska B , Dours-Zimmermann M T , Odermatt B F , Briner J , Heitz P U , Zimmermann D R
Published: April 2011

We studied the distribution of the large hyaluronan-binding proteoglycan versican (also known as PG-M) in human adult tissues using affinity-purified polyclonal antibodies that recognize the core protein of the prominent versican splice variants VO and V1. Versican was present in the loose connective tissues of various organs and was often associated with the elastic fiber network. Furthermore, it was localized in most smooth muscle tissues and in fibrous and elastic cartilage. Versican staining was also noted in the central and peripheral nervous system, in the basal layer of the epidermis, and on the luminal surface of some glandular epithelia. In blood vessels, versican was present in all three wall layers of veins and elastic arteries. In muscular arteries the immunoreactivity was normally restricted to the tunica adventitia. However, it appeared in the media and the split elastica interna of atherosclerotically transformed vessel walls. Our survey of the distribution of versican in normal human tissues now forms the basis for extended studies of potentially aberrant versican expression during pathogenic processes.
Immunocytochemical and cytochemical demonstration of a novel selective lysosomal pathway (SLP) of secretion in the exocrine pancreas.
Authors: Grondin G , Beaudoin A R
Published: April 2011

The intracellular distributions of lysosomal and zymogen granule (ZG) membrane proteins were analyzed in the pancreas exocrine acinar cell by cytochemical and immunocytochemical approaches. A strong signal was observed with acid phosphatase (AcPase) in the trans-Golgi network and condensing vacuoles, whereas mature ZG and acinar lumina were devoid of any detectable reaction. The enzyme appears to exit from the regulated pathway by a shedding process during conversion of condensing vacuoles to mature granules. Trimetaphosphatase (TMPase) shows no reaction in the Golgi apparatus and condensing vacuole but is present in immature granules. The exit from the regulated pathway appears to occur at a later stage of the ZG maturation process. A third lysosomal enzyme, nicotinamide adenine dinucleotide phosphohydrolase (NADPase), was found in the median cisterna of the Golgi stack, was undetectable in condensing vacuoles and ZG, but produced a strong signal in the acinar lumen. Our observations show that only one type of intermediate organelle can explain the transport of that enzyme from the Golgi apparatus to the acinar lumen, and it is represented by a subpopulation of lysosomal bodies (LBs) highly reactive for this enzyme. In parallel, immunocytochemical observations with specific antibodies to GP2, a major protein component of the ZG membrane, have confirmed that most of the GP2 molecules in the acinar lumen do not derive from the ZG compartment but rather are from a subpopulation of highly reactive lysosomal structures. Because both NADPase and GP2 co-localize in specific lysosomal structures, and because these LBs are extruded in the acinar lumen, we conclude that this subpopulation of LBs is involved in selective transport of some lysosomal enzymes from the Golgi apparatus to the acinar lumen. This selective lysosomal pathway of secretion can explain the kinetics of GP2 transport and release from the acinar cell that cannot be explained either by the constitutive or the regulated pathway of secretion.
Organ distribution of aminopeptidase A and dipeptidyl peptidase IV in normal mice.
Authors: Mentzel S , Dijkman H B , Van Son J P , Koene R A , Assmann K J
Published: April 2011

The hydrolases aminopeptidase A and dipeptidyl peptidase IV, both present in the kidney on the brush borders of the proximal tubule epithelial cells and podocytes, are involved in the induction of experimental membranous glomerulonephritis in the mouse. However, little is known about their (co)distribution in other tissues and their function in health and disease. A detailed insight into the localization of these two enzymes is a prerequisite to elucidation of their function. Therefore, we investigated the presence and co-distribution of aminopeptidase A and dipeptidyl peptidase IV by immunohistology with two different rat monoclonal antibodies, the specificity of which was determined by an immunodepletion technique. In addition, the molecular weight of the hydrolases; was analyzed by SDS-PAGE after isolation by solid-phase immunoprecipitation from glomeruli, renal brush borders, and thymus. Both hydrolases showed different molecular weights in renal corpuscle, renal brush borders, and thymic cells. A widespread organ distribution of the two hydrolases was observed, with co-localization in kidney, liver, small intestine, thymus, brain, spleen, and lymph nodes, either on the same cells or on different cells in the same organ. This distribution and partial co-localization suggests that the two hydrolases, acting either alone or in concert, have a role in many diverse biological processes.
Mapping of corticotropic cells in the normal human pituitary.
Authors: Trouillas J , Guigard M P , Fonlupt P , Souchier C , Girod C
Published: April 2011

We accomplished the first mapping of corticotropic cells in the whole human adult pituitary. Corticotropic cells were identified by immunocytochemistry (ICC) and quantified by image analysis on 12 pituitaries obtained from people who had died suddenly. An overall view of each pituitary was given by 15-21 sections (mean 18 sections) at 300-micron intervals on six slides. Each section was systematically treated by indirect immunoperoxidase using an anti-ACTH[17-39] polyclonal antiserum. All the measures were done with a x 6.3 objective lens, each field (0. 5 mm2) being considered as the unit area. The mean pituitary density (surface of labeled cells/total surface) of corticotropic cells (9.5 +/- 3.0% per 0. 5 mm2) is significantly higher in men (11.5 +/- 5.1%) than in women (7.0 +/- 1.3%). This difference is due to an inverse relationship between the corticotropic cell density and the weight of the pituitary, which is higher in women than in men. The mean diameter of corticotropic cells is 14.9 micron and their total number per pituitary is approximately 10(7) cells. We confirmed that the spatial distribution of corticotropic cells is nonuniform: they are mainly distributed in the anteromedian part of the anterior lobe. In addition, our results demonstrated that the inferior part of the pituitary contained three times more corticotropic cells than the superior part (mean density 18.0% vs 6.0%) and the anterior part twice as many as the posterior part (mean density 12.3% vs 6.8%). On the horizontal plane, the pituitary was divided into eight zones, in which the mean of area was 2.5-21.0%. The maximal cell density may reach 40-60%. The use of this map should help the pathologist to recognize if there is corticotropic hyperplasia in a small pituitary fragment surgically removed from a patient with Cushing's disease. On the basis of this study, we put forward some criteria for diagnosing corticotropic hyperplasia.
Histochemistry to detect Helix pomatia lectin binding in breast cancer: methodology makes a difference.
Authors: Brooks S A , Lymboura M , Schumacher U , Leathem A J
Published: April 2011

A number of studies have shown that altered cellular glycosylation, as detected by binding of Helix pomatia lectin to paraffin sections, is associated with metastatic disease and consequent poor patient prognosis in breast and other cancers. In a 24-year retrospective study, sections of 373 primary breast cancers were stained for binding of the lectin using two different histochemical techniques: a direct method (using peroxidase-conjugated lectin) and an indirect method (using native, unconjugated lectin). Similar percentages of cases were positive (79%) and negative (21%) for lectin binding with either technique, but there was enormous inconsistency when individual cases were examined. A total of 38/373 (10.2%) cases that were negative by the indirect method were positive by the direct method, and 37/373 (9.9%) cases that were negative by the direct method were positive by the indirect method. Life tables calculated for lectin staining vs nonstaining cases showed a very strong correlation between lectin binding and long-term survival (p < 0.0001) when staining was performed by the indirect method, but only very weak correlation with prognosis (p < 0.03, borderline significance) when the direct technique was employed. SDS-PAGE revealed that there were differences in breast cancer glycoproteins recognized by native lectin and peroxidase-conjugated lectin immobilized on Sepharose 4B affinity beads. Helix pomatia lectin binding appears to be an intriguing and potentially valuable marker of biological behavior in breast cancer. This study emphasizes the importance of selecting an appropriate immunohistochemical technique for its visualization.
Dihydrorhodamine 123 identifies impaired mitochondrial respiratory chain function in cultured cells harboring mitochondrial DNA mutations.
Authors: Sobreira C , Davidson M , King M P , Miranda A F
Published: April 2011

Several human diseases have been found to be caused by mitochondrial DNA (mtDNA) mutations. Pathogenic mutated (mut) mtDNAs are usually "heteroplasmic," coexisting intracellularly with wild-type (wt) mtDNAs. For some mtDNA mutations, cells have normal levels of respiratory chain function unless the percentage of mut-mtDNA is very high. Although progress in understanding the molecular basis of mitochondrial diseases has been remarkable, the heterogeneity of mut-mtDNA distribution, even among cells of the same tissue, makes it difficult to clearly delineate the relationships between mtDNA mutations, gene dosage, and clinical phenotypes. In a search for screening methods for identifying cultured cells with deficient mitochondrial function, we incubated living cells harboring mut-mtDNAs with dihydrorhodamine 123 (DHR123), an uncharged, nonfluorescent agent that can be converted by oxidation to the fluorescent laser dye rhodamine 123 (R123). Bright mitochondrial staining was observed in cells that respired normally. Fluorescence was significantly reduced in cells with mitochondrial respiratory chain dysfunction resulting from very high levels of mut-mtDNAs. The data show that DHR123 is useful for assessing mitochondrial function in single cells, and can be used for isolating viable, respiratory chain-deficient cells from heterogeneous cultures.
Detection and spatial distribution of IL-2 receptors on mouse T-lymphocytes by immunogold-labeled ligands.
Authors: Breitfeld O , Kühlcke K , Lother H , Hohenberg H , Mannweiler K , Rutter G
Published: April 2011

To identify the plasma membrane (PM) structures implicated in T-cell activation, we studied the distribution of interleukin-2 receptors (IL-2R) and the surface topography of lymphocytes by affinity labeling in electron microscopy (EM). In particular, we analyzed the distribution of the IL-2R alpha-chain on CTLL-2 cells (a murine cytotoxic T-cell lymphoma line). Some of our experiments were extended to the functionally and morphologically distinct cell line EL4 (a routine helper T-cell lymphoma line). As affinity ligands we used a rat monoclonal antibody (clone 7D4) reactive with the routine alpha-chain of IL-2R and recombinant mouse IL-2 (rIL-2). The distribution of IL-2R was visualized on the cell surface by ligands coupled to colloidal gold particles of different sizes. Unfixed cells were labeled with gold probes and attached to concanavalin A (ConA)-pretreated coverslips. Subsequently, the cells were prepared for EM. Examination of ultrathin sections and large surface replicas revealed a high degree of variability in cell morphology and in the density of the randomly distributed gold-labeled ligands among CTLL cells. According to their typical appearance, lymphocytes with strong receptor expression can be easily identified within the cell population. In contrast, the label on many mitogen-activated EL4 cells showed a cap-like polar distribution. The results suggest the existence of diverse distribution patterns of IL-2R on CTLL and EL4 cells. These differences are believed to reflect the different physiological roles played by T-cell subsets in the immune system.
Expression of the pituitary transcription factor GHF-1/PIT-1 in cell types of the adult porcine adenohypophysis.
Authors: Malagon M M , Garrido J C , Dieulois C , Hera C , Castrillo J L , Dobado-Berrios P M , Gracia-Navarro F
Published: April 2011

We describe the expression of the transcription factor GHF-1/PIT-1 in adult porcine adenohypophysis by a nonradioactive in situ hybridization (ISH) method using a digoxigenin-labeled cDNA probe corresponding to the entire coding region of rat GHF-1. GHF-1 transcripts were found in 71.7% of adenohypophyseal cells. We also report the simultaneous detection of GHF-1 mRNA and pituitary hormones by combined ISH and immunocytochemistry (IC) in dispersed adenohypophyseal cells, detected with an alkaline phosphatase-NBT/BCIP technique and with an immunoperoxidase-3-amino-9-ethylcarbazole (AEC) method, respectively. The combination of the two techniques neither abolished nor diminished their sensitivity or specificity. GHF-1 is expressed in all five of the cell types in the adult porcine adenohypophysis, showing that this method is suitable for simultaneous detection of transcripts and proteins at the single-cell level.
A quantitative luminescence assay for nonradioactive nucleic acid probes.
Authors: Didenko V V , Hornsby P J
Published: April 2011

In histochemical work with digoxigenin- or biotin-labeled nucleic acid probes, reproducibility of in situ hybridization depends on accurate measurement of the amount of non-radioactive label being used. We describe a rapid and sensitive assay for nonradioactive label incorporated into nucleic acids employing a luminogenic substrate for alkaline phosphatase, CSPD (disodium 3-(4-methoxyspirol¿1,2-dioxetane-3,2'-(5'-chloro)tricyclo [3.3.1.1(3,7)]decan¿-4-yl)phenyl phosphate). An alkaline phosphatase-antibody conjugate was bound to digoxigenin-labeled nucleic acids spotted on nylon membranes. Light emission from the reaction of the bound alkaline phosphatase with CSPD was measured with a luminometer. This method allows an accurate determination of digoxigenin incorporated into nucleic acid probes in the range of 0.5-500 fmol of nonradioactive label.
Comparison of 35S and chemiluminescence for HPV in situ hybridization in carcinoma cell lines and on human cervical intraepithelial neoplasia.
Authors: Lorimier P , Lamarcq L , Negoescu A , Robert C , Labat-Moleur F , Gras-Chappuis F , Durrant I , Brambilla E
Published: April 2011

For in situ hybridization (ISH), development of sensitive, nontoxic alternatives to the use of radioactivity is a constant concern. In this trend, and close to chromogenes and fluorophores, chemiluminescence appears an attractive method. A first positive experience in immunocytochemistry and in ISH, by using the enhanced luminol as luminogene substrate for horseradish peroxidase (HRP) led us to compare the sensitivity of 35S autoradiography and chemiluminescence. For this purpose, we used three human carcinoma cell lines, CaSki [400-600 copies of human papilloma virus (HPV) 16], HeLa (10-50 copies of HPV 18), and SiHa (1-5 copies of HPV 16), and 40 biopsy specimens of human cervical preneoplastic and neoplastic lesions. We performed ISH by using HPV cDNA biotin-labeled probes, detected by a two-step immunocytochemical reaction, the secondary antibodies being either 35S-labeled for autoradiography or HRP-labeled for chemiluminescence. An intensified CCD camera allowed acquisition of the luminescent signal. After only 10 min of photon accumulation, on cell line smears as well as on serial tissue sections, chemiluminescence gave comparable results to those obtained by a 3-week exposure for 35S autoradiography. A quantitative approach on cervical biopsy specimens confirmed this similar level of sensitivity by measuring the area of 35S- or chemiluminescence-stained nuclei. Our results indicate that chemiluminescence is a credible and perfectible alternative to radioisotopes for in situ detection of nucleic acids by hybridization.
Expression of thyroid transcription factor-1(TTF-1) in fetal and neonatal human lung.
Authors: Stahlman M T , Gray M E , Whitsett J A
Published: April 2011

We assessed the immunohistochemical localization of thyroid transcription factor-1 (TTF-1) in the lungs of 24 human fetuses (11-23 weeks), three infants without pulmonary pathology (36-42 weeks), and 24 infants (2 days-6.5 months) with hyaline membrane disease (HMD) or bronchopulmonary dysplasia (BPD). TTF-1 was detected in fetal lung epithelial cell nuclei by 11 weeks' gestation. Budding tips of terminal airways had prominently labeled nuclei. By 17 weeks, labeling was present in scattered nonciliated columnar and cuboidal cells. Throughout gestation, TTF-1 nuclear staining was prominent in airways abutting pleural, peribronchial, or perivascular connective tissue, being less prominent in centers of lobules. By 23 weeks, many cells in cuboidal but not columnar cell-lined airways had labeled nuclei. At term, TTF-1 was detected primarily in Type II epithelial cells. In HMD with alveolar hemorrhage, edema, or airway collapse, little or no TTF-1 was present except in open terminal airways. In BDP lungs, TTF-1 was absent in areas of alveolar collapse or infection, being present in regenerating open airways. The temporal-spatial distribution of TTF-1, in general, follows patterns of distribution of surfactant protein-B in developing and pathological lungs, consistent with its role in the regulation of epithelial cell gene expression in the lung.