Journal of Histochemistry & Cytochemistry

Previous freeze-fracture experiments using either glutaraldehyde-fixed and cryoprotected specimens or unfixed rapid-frozen samples led to the proposal that cylindrical strands of the tight junction (TJ) observed in freeze-fracture preparations are inverted cylindrical micelles made up of membrane lipids and, possibly, membrane proteins. However, no one has yet been able to directly label the structural fibrils of the TJ. To test the hypothesis that TJ strands observed on freeze-fracture preparations are composed at least partially of lipids, we have combined the phospholipase A2-gold and the fracture-label techniques for localization of phospholipids. Phospholipase A2, purified from bee venom, was adsorbed on gold particles and used for specific labeling of its substrate. Phospholipase A2-colloidal gold (PLA2-CG) complex was applied to freeze-fractured preparations of rat exocrine pancreatic cells and testicular Sertoli cells, both of which are known to have extensive TJ complexes on their plasma membranes. Fracture-label replicas of exocrine pancreatic cells revealed specific association of gold particles with TJ fibrils on the protoplasmic fracture-face of the plasma membrane. The majority of these gold particles were observed either directly on the top of the TJ fibrils or adjacent to these cylindrical structures. A high density of PLA2-CG labeling was also observed over the complementary exoplasmic fracture-face of the TJ complex. This intimate association of PLA2-CG labeling with the TJ is particularly evident in the Sertoli cell plasma membrane, where rows of gold particles were observed to be superimposed on parallel arrays of cylindrical strands of the TJ complex. The present findings provide direct cytochemical evidence to support the hypothesis that cylindrical TJ strands observed in freeze-fracture preparations contain phospholipids.

A new method is described for locating RNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing polyadenylate nucleotidyl transferase (PnT) and biotinylated ATP. The labeled nucleotides bound to RNA at the surface of the ultra-thin sections were than visualized by an indirect immunogold labeling technique. The resulting labeling pattern was dependent on the presence of divalent cations in the PnT medium. The method revealed with great precision the specific RNA-containing structures within Ehrlich tumor cells. The method is applicable to Epon sections. However, the labeling intensity varies according to the fixation used. Best results were obtained on acetylated cell sections. The method can be combined with EDTA regressive staining. The in situ PnT method provides a very useful tool for pinpointing the precise location of RNA within biological material at the ultrastructural level.

Xanthine oxidoreductase exists in two functionally distinct forms. Under normal conditions, the larger part of the enzyme occurs as an NAD(+)-dependent dehydrogenase form which produces NADH and urate. The dehydrogenase can be transformed under various (patho)physiological conditions to an oxygen-dependent oxidase form which produces oxygen radicals and/or hydrogen peroxide and urate. Tetrazolium salts are used to demonstrate the total activity of both the dehydrogenase and the oxidase form of the enzyme. We have developed a procedure to detect the oxidase form only in unfixed cryostat sections with the use of cerium on the basis of the semipermeable membrane technique. The incubation medium contained hypoxanthine as substrate, cerium ions, and sodium azide to inhibit catalase and peroxidase activity. In a second-step reaction, diaminobenzidine was polymerized in the presence of cobalt ions by decomposition of cerium perhydroxide. Large amounts of final reaction product were found in milk droplets in the acini of lactating bovine mammary gland, whereas milk-secreting epithelial cells contained hardly any final reaction product. In rat duodenum, enzyme activity was found in the cytoplasm of enterocytes and goblet cells but not in the mucus. Control reactions performed in the absence of substrate or in the presence of substrate and allopurinol, a specific inhibitor of xanthine oxidase, were completely negative in both tissues, with the exception of polymorphonuclear leukocytes in the lamina propria of duodenum. The positive nonspecific reaction in these cells was caused by myeloperoxidase activity. We conclude that the present method is specific for the detection of xanthine oxidase activity. Moreover, conversion of the dehydrogenase form into the oxidase form can be prevented by omission of chemical fixation of the tissue in the present procedure.

We investigated the immunohistochemical localization of androgen receptor (AR) using a polyclonal antibody for 55 KD recombinant human AR in human tissues fixed with 4% paraformaldehyde solution and embedded in paraffin. Immunoreactive AR was restricted to the nuclei of various tissues. Among the well-known androgen target organs, secretory cells and basal cells of the prostate, spermatogonia, spermatocytes, Sertoli cells and Leydig cells of the testis, epithelial cells of the rete testis, fibroblasts in the whole organ, squamous cells, sweat gland and hair follicle cells of the skin, and hepatocytes of the liver were positive for AR. In addition, smooth muscle cells of the prostate, uterus, urinary bladder, gastrointestinal tract, arteries, and arterioles were strongly immunoreactive for AR. Cardiac muscle and striated muscle of psoas were positive for AR. Acinar cells, ductal cells, and myoepithelial cells of the breast, endocervical and endometrial cells of the uterus, cyto- and syncytiotrophoblast of the chorionic villi, and tubules of the kidney were also positive for AR. Most FSH, LH, and some GH endocrine cells in the anterior and posterior lobes of the pituitary gland, follicular cells of the thyroid gland, and adrenocortical cells were positive for AR. Cells immunoreactive for AR were ubiquitously distributed throughout the entire body. The present study demonstrated the diversity of androgen effects on many target tissues.

We investigated the spatiotemporal localization of collagen Type I, II, and X mRNAs in the subcutaneously grafted chick periosteum by in situ hybridization. Five days after transplantation, we noted three types of histological findings in the grated tissue. (a) Developing trabecular bone exhibited proliferation of spindle-shaped fibroblastic cells and polygonal osteoblasts with moderate signals for collagen Type I mRNA. (b) Developing cartilage contained ovoid chondrocytes with a moderate level of both collagen Type I and II mRNAs. Differentiating chondrocytes with increased collagen Type X mRNA developed during the course of endochondral ossification. (c) An atypical mass of cartilage weakly stained with alcian blue was composed of a large number of non-hypertrophic chondrocytes exhibiting high signals for collagen Type X mRNA. At Day 9, we observed the typical histological features of both membranous and endochondral ossification. However, sparsely distributed chondrocytes with high signals for collagen Type X mRNA were also demonstrated in osteoid and/or woven bone. The phenotype of chondrocytes showing rapid expression of collagen Type X gene derived from grafted periosteum seems to participate in the important role of endochondral bone formation in the early stage of fracture repair.

A nine amino-acid peptide derived from the beta 1-subunit of the bovine GABAA receptor was used for immunization of mice and subsequent production of monoclonal antibodies (MAb). In view of the later immunocytochemical application of the MAb to sections of cat visual cortex, the MAb were characterized on similar tissue. The GABAA receptor was isolated by affinity chromatography of protein material obtained from cat cortical gray matter. The antibodies were characterized by SDS-PAGE, followed by immunoblotting and immunoadsorption. Immunocytochemical staining with the MAb revealed labeled cells throughout all layers of the cat visual cortex as well as within the white matter. The morphology of most stained cells in the white matter and of some cells in the cortical gray matter closely resembled that of astrocytes. Double immunocytochemical staining with an antiserum against glial fibrillary acidic protein (GFAP), followed by light microscopic examination, indeed confirmed that astrocytes in both white and gray matter in the cat cortex possess GABAA receptors.

We developed a technique to map the distribution of selected metabolites in the growth cartilage in situ using luciferase-NAD(P)H:FMN oxidoreductase. Chick tibial epiphyses were freeze-trapped, sectioned, and freeze-dried. For evaluating lactate, luciferase was suspended in a buffer containing polyvinylalcohol, gelatin, NAD, FMN, and lactic dehydrogenase (LDH). The buffer was frozen into a layer 800 microns thick and placed in contact with the tissue section. The temperature of the frozen reagent mixture was then allowed to increase; the emitted light was focused through a photographic lens and collected on film. We found that lactate was synthesized by cells in all regions of the growth plate. The highest concentration of the metabolite was observed in the calcified hypertrophic region. Substantial levels of lactate were also present in articular cartilage. By modifying the composition of the buffer solution, we were able to map the distribution of glucose and glucose-6-phosphate and the activity of LDH. Maximal levels of each of the three components were present in hypertrophic cartilage. Chemical analysis of the tissue section confirmed the luminographic studies and provided further evidence that there was reliance on glycolytic metabolism in terminally differentiated chondrocytes. Use of enzyme couples similar to those described above should permit the technique to be used to study most, if not all, of the major metabolic components of cartilage.

Nerve growth factor (NGF) is a protein highly expressed in the male mouse submandibular gland. We have applied a non-radioactive in situ hybridization method using digoxigenin-labeled NGF oligonucleotides, and have found the highest amounts of NGF mRNA in the secretory striated ducts of the male mouse submandibular gland. Scattered strongly positive cells were found in male mouse sublingual glands. Weakly labeled cells were seen in female mouse and in male rat submandibular gland striated duct cells. Using 33P as an alternative to 32P and 35S, we demonstrated a 1.3 KB NGF mRNA in salivary glands of male mice by Northern blot hybridization. Using 33P we detected NGF mRNA in male mouse submandibular glands by in situ hybridization but with a signal that, compared with the non-radioactive method, had a very low resolution. Castration of male mice almost abolished both the 1.3 KB NGF mRNA seen with Northern blots and the NGF mRNA labeling in submandibular glands 4 weeks after the operation, whereas levels were increased 6 hr and 2 days after sympathectomy. We conclude that hybridization with digoxigenin-labeled NGF oligonucleotides is a good tool to study the expression and regulation of NGF mRNA in male mouse submandibular glands.

In an attempt to localize the guanine nucleotide binding protein Gs alpha by immunocytochemistry, we synthetized peptides corresponding to several stretches of residues deduced from the published cDNA sequence of Gs alpha and raised antibodies against them. Among the peptides, one corresponding to residues 367-381 elicited antibodies that immunocytochemically detected, at the optical level, what appeared to be vimentin in several cells and tissues. Studies at the ultrastructural level confirmed this observation and also showed weak staining of some areas of plasma membranes of glial and nerve cells. Analysis by Western blots of rat brain subcellular fractions indicated that: (a) the protein stained by the anti-peptide antibodies was associated with the cytoskeleton; and (b) it was not vimentin but a protein of higher molecular weight, 65 KD. We accordingly suggest that the Gs alpha-derived peptide elicited two types of antibodies, one recognizing Gs alpha in fixed tissues, the other recognizing an epitope located in a vimentin-associated protein. This study emphasizes the caution that is needed when conclusions are drawn on the basis of immunocytochemical studies using antipeptide antibodies.

In this study we compared the sensitivity of immunocytochemical procedures, using conventional and time-resolved fluorescent dyes, in a model system consisting of paraformaldehyde-fixed human lymphocytes. The lymphocytes were stained for the presence of the CD4 epitope by indirect immunofluorescence using FITC as label or by using time-resolved luminescent immunophosphors. These immunophosphors were primarily developed for use under time-resolved fluorescence conditions, but they are also very well suited for use in conventional fluorescence microscopes. The differently labeled cells were first examined visually with a conventional fluorescence microscope in a double-blind study. The fluorescence was also measured with a CCD camera mounted on a specially constructed time-resolved fluorescence microscope which allows the suppression of the fast decaying fluorescence, thereby permitting visualization of the specific, slowing decaying luminescence of the phosphor label. With this microscope FITC and immunophosphor labeled lymphocytes were compared under normal conditions (i.e., continuous excitation) and under conditions of time-resolved registration. Conventional fluorescence microscopy revealed a better sensitivity in favor of the phosphor conjugates. This difference became more prominent when the preparations were quantitatively assessed with the CCD-time-resolved microscope. Time-resolved microscopy permitted a suppression of fast decaying fluorescence better than 1 to 10(6).

Kidneys of copper-loaded rats were investigated immunohistochemically with a directly peroxidase-conjugated monoclonal antibody against metallothionein (MT). By means of an image analyzing system the area and the staining intensity of MT immunoreactivity in the proximal convoluted tubule (PCT) cells were determined. In the present study we compared the data obtained by image analysis with analytically determined tissue copper and MT concentrations of rats fed a high-copper diet (1 g/kg) for 16 weeks and sacrificed sequentially during this period. Our results provide evidence that the area of MT immunoreactivity correlates significantly with tissue copper and MT concentrations. Both the copper and MT concentrations in kidney rose to a maximum at 8 weeks and remained constant thereafter. The observed rise in the staining intensity of MT in PCT cells to a maximum at 6 weeks, which subsequently declined, suggests a continuing redistribution of copper and MT in the kidneys even after a maximum of concentration copper and MT is reached in the tissue.

This study characterized histochemically three fast fiber types (IIB, IID, IIA) in skeletal muscles of mouse, rat, and rabbit, with special reference to fiber types IIB and IID. The results are complemented by biochemical analyses of myosin heavy chain composition in these muscles. Fiber type delineation is based on various methods for mATPase staining with pre-incubations and assays under different conditions. In rat and mouse, IIB and IID fibers can be best distinguished according to their different mATPase stabilities towards formaldehyde and alkaline pH. In rabbit, the method of Matoba and Gollnick using acid pre-incubation provided best and most reproducible results. In addition to their different mATPase stabilities, the three fast fiber types differ with regard to their oxidative capacities and cross-sectional fiber areas in the three species. In general, Type IIB fibers are the largest and least oxidative, Type IIA fibers the smallest and most oxidative, and Type IID fibers intermediate. In rabbit, Type IID fibers are the predominant fast fiber population in extensor digitorum longus, psoas, and tibialis anterior muscles. As judged from histochemistry, these muscles of rabbit do not contain pure Type IIB fibers. This is in accordance with biochemical results that show the HCIId to form the majority of the myosin heavy chain complement expressed in these muscles. On the other hand, IIB fibers are numerous in rabbit adductor magnus, gastrocnemius, and vastus lateralis muscles. Similarly, appreciable amounts of myosin heavy chain HCIIb are found in the three latter muscles of rabbit.

Dry and rehydrated birch pollen grains were anhydrously fixed and double immunogold-labeled for the presence of two allergens, Bet v I major allergen (17 KD) and profilin (14 KD). In dry pollen grains, both allergens are found exclusively inside the cytoplasm. In pollen grains rehydrated for 1 min, the cytoplasm is partially devoid of the two allergens, whereas the pollen wall and the germination aperture are specifically labeled. Pollen grains rehydrated for 5 min are largely free of the two allergens. In immunoblot experiments, both allergens could be detected in the aqueous supernatants of rehydrated pollen samples within 5 min. The results obtained by both methods show the high solubility of both proteins. This makes them readily available to the immune system and characterizes them as potent allergens. Moreover, the solubilization of profilin might indicate a dissociation of the profilin-actin complex at the very first stage of pollen germination, which could favor formation of the cytoskeleton and pollen tube growth.

We studied the location of a membrane-bound carbonic anhydrase (CA IV) in the human male reproductive tract using a specific antiserum to human CA IV in conjunction with immunoblotting, immunoperoxidase, and immunofluorescence techniques. The microvilli and apical plasma membrane of the epithelial cells and the subepithelial smooth muscle layer of the epididymis, ductus deferens, and ampulla of the ductus deferens showed specific staining for CA IV. The epithelial cells of the prostate and seminal vesicle failed to stain for CA IV, however, whereas the subepithelial smooth muscle layer showed positive staining. No specific staining for CA II was seen in the epithelium of the epididymal duct or the proximal ductus deferens. The presence of CA IV in the epididymis was confirmed by immunoblotting, which revealed 35 KD and 33 KD polypeptides. The results show that the microvilli and the apical plasma membrane of the lining epithelium of the epididymal duct, ductus deferens, and ampulla of the ductus deferens contain the membrane-bound carbonic anhydrase isoenzyme IV. The presence of the enzyme in the epithelium of the epididymis and ductus deferens is probably linked to the acidification of the epididymal fluid that prevents premature sperm activation. Its physiological role in the smooth muscle cells remains to be elucidated.

We used an immunohistochemical method for the inner mitochondrial membrane uncoupling protein (UCP) to examine whether human brown adipose tissue UCP could be detected by an anti-rat UCP antibody. Samples of human brown adipose tissue were obtained at medicolegal autopsies. Fat tissue was excised from around the common carotid arteries and in the subscapular region and from around the thoracic aorta. The subjects were either known alcohol consumers, in which thermogenically active brown adipose tissue (BAT) is often found, or victims of sudden infant death syndrome (SID). UCP was detected in all the cases examined, even when the post-mortem time from death to autopsy reached several days, but the intensity of the immunostaining was variable. Intense staining was observed in three cases with a post-mortem time under 24 hr, but in the SID cases a strong positive staining was seen even with a post-mortem delay of 4 days. These results show that an anti-rat UCP antibody can be used for immunohistochemical detection of UCP in human brown adipose tissue and that it provides a useful method for distinguishing between white and brown fat in paraffin-embedded samples. It can be used to detect UCP in the BAT of obese and diabetic individuals and probably also in the histopathological diagnosis of brown adipose tissue lipoma, known as hibernoma.

We have developed a new, easy, and more rapid method for DNA preparation, which avoids contamination. With this method, manual surgical blade scrapings from precisely targeted areas of paraffin block surfaces, without microtome cutting, were used to obtain tissues from 10 different neoplasms. Our results indicate the feasibility of DNA extraction from the scraped paraffin tissue for molecular genetic analysis. We applied this technique successfully to screen for the presence of human papillomavirus using the polymerase chain reaction (PCR) procedure in cases of endocervical, esophageal, and nasopharyngeal carcinomas, and to examine the expression of p53 gene from prostate and gastric adenocarcinomas. We conclude that this procedure is also suitable for purification of PCR products in analysis of the mutation or loss of allelic genes by Bstu I endonuclease digestion.

We describe in detail a simple method for flat-embedding that can be subsequently used in correlative light and electron microscopic studies. The method can be applied to any material suitable for electron microscopy and is especially useful for study of the synaptology and ultrastructural characteristics of immunocytochemically or morphologically identified neurons or their processes. We present here an example to show how accurately one can delineate the fine details of a complex axonal arborization impregnated with the Golgi method in the mouse cerebral cortex. Golgi-impregnated sections to be studied at the electron microscopic level are osmicated, dehydrated, infiltrated with Araldite resin, flat-embedded, and identified cells or processes photographed. Serial semi-thin sections (1-2 microns thick) are then cut with an ultramicrotome, examined with the light microscope, and the elements rephotographed. Selected semi-thin sections are then resectioned on the ultramicrotome at 60-70 nm and examined electron microscopically. This method allows the systematic and accurate localization of stained cells and processes throughout the successive steps of the procedure.

In situ hybridization histochemistry (ISH) using cRNA probes (riboprobes) has become a powerful technique for the examination of gene expression in tissue sections. The construction of plasmid templates for the synthesis of riboprobes with phage RNA polymerases is often a difficult and time-consuming step. We have therefore developed a rapid, efficient, and flexible method to generate totally artificial riboprobe templates by the polymerase chain reaction (PCR). We have made riboprobe templates using self-priming oligonucleotide primers spanning 146 BP of the 3' end of the human cytokeratin 1 (K1) gene coding region flanked by T7 and T3 promoters. These PCR-derived riboprobe templates were used to synthesize 35S-labeled anti-sense riboprobes as well as sense riboprobes as negative controls. The riboprobes were then applied in ISH to human skin sections made from routinely fixed and paraffin-embedded clinical biopsy material. Consistent with published results, we observed strong expression of K1 mRNA in the suprabasal cell layers of the epidermis but only weak to undetectable signals in the basal and cornified cell layers and in the dermis. With this experimental procedure we see no decrease in probe efficiency or quality compared to conventional methods. The use of PCR-derived riboprobe templates for ISH makes it possible to detect expression of any desired gene of known sequence rapidly and efficiently.

Fluoro-Ruby, the fluorescent tetramethylrhodamine-dextran-amine used to demonstrate anterograde axon transport, has been successfully photoconverted and subsequently localized by electron microscopy. The photoconversion was accomplished by irradiating the tissue with green light while bathing it in a solution containing DAB. The tissue could then be examined by brightfield microscopy or processed for conventional electron microscopy. Potential advantages of the technique include greater permanence and contrast at the light microscopic level and the ability to resolve synaptic connectivity at the electron microscopic level.