Journal of Histochemistry & Cytochemistry

We review here the oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase (G6PDH) activity to detect cancer cells. This inexpensive and rapid assay can be performed within half an hour. Discrimination between cancerous and noncancerous cells is based on a combination of elevated G6PDH activity, decreased super-oxide dismutase (SOD) activity, and decreased lipid peroxidation in cancer cells. The test discriminates between adenomas and carcinomas of the colon with a certainty of >80% and has a high prognostic value for survival of colon cancer patients. Pancreatitis and pancreatic cancer are discriminated with a certainty of 100%. Therefore, the test can be applied by pathologists to provide additional information in difficult cases of diagnosis of cancer and for prognosis.

To investigate the pattern of expression of the three calmodulin (CaM) genes by in situ hybridization, gene-specific [³⁵ S]-cRNA probes complementary to the multiple CaM mRNAs were hybridized in rat brain sections and subsequently detected by quantitative film or high-resolution nuclear emulsion autoradiography. A widespread and differential area-specific distribution of the CaM mRNAs was detected. The expression patterns corresponding to the three CaM genes differed most considerably in the olfactory bulb, the cerebral and cerebellar cortices, the diagonal band, the suprachiasmatic and medial habenular nuclei, and the hippocampus. Moreover, the significantly higher CaM I and CaM III mRNA copy numbers than that of CaM II in the molecular layers of certain brain areas revealed a differential dendritic targeting of these mRNAs. The results indicate a differential pattern of distribution of the multiple CaM mRNAs at two levels of cellular organization in the brain: (a) region-specific expression and (b) specific intracellular targeting. A precise and gene-specific regulation of synthesis and distribution of CaM mRNAs therefore exists under physiological conditions in the rat brain.

In freshly isolated rat hepatocyte multiplets, Ca²⁺ signals in response to vasopressin are highly organized. In this study we used specific probes to visualize, by fluorescence and confocal microscopy, the main signaling molecules involved in vasopressin-mediated Ca²⁺ responses. V1a receptors were detected with a novel fluorescent antagonist, Rhm⁸ -PVA. The Gαq/Gα11, PLCβ₃, PIP₂, and InsP₃ receptors were detected with specific antibodies. V1a vasopressin receptors and PIP₂ were associated with the basolateral membrane and were not detected in the bile canalicular domain. Gαq/Gα11, PLCβ₃, and InsP₃ receptors were associated with the basolateral membrane and also with other intracellular structures. We used double labeling, Western blotting, and drugs (cytochalasin D, colchicine) known to disorganize the cytoskeleton to demonstrate the partial co-localization of Gαq/Gα11 with F-actin.

We investigated the histochemical characteristics of mast cell tryptase in different mouse tissues. By use of peptide substrates, tryptase activity could be demonstrated in unfixed connective tissue mast cells in different tissues, including the stomach. Tryptase activity was better localized after aldehyde fixation and frozen sectioning, and under such conditions was also demonstrated in mucosal mast cells of the stomach but not in those of the gut mucosa. Double staining by enzyme histochemistry followed by toluidine blue indicated that the tryptase activity was present only in mast cells and that all mast cells in the stomach mucosa contained the enzyme. The peptide substrates z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthlyamide, which are substrates of choice for demonstrating tryptase in other species, were most effective for demonstrating mouse tryptase. The use of protease inhibitors further indicated that activity present in all mast cells was tryptase. Safranin O did not stain stomach mucosal mast cells, suggesting that the tryptase present in these cells was active in the absence of heparin sulfate proteoglycan.

The enzyme PAM is required for activation of many peptide hormones. In adult mouse lung, immunostaining for PAM was located in Clara cells, which constitute most of the epithelial cells of the mouse bronchial/bronchiolar tree. Immunoreactivity appeared for the first time in the epithelium on gestational Day 16, being slight and mostly restricted to the apical cytoplasm. As the lung developed, the labeling became gradually stronger and extended throughout the cell. Smooth muscle of airways and blood vessels, and some parenchymal cells, probably macrophages, also showed PAM immunoreactivity. Of the two enzymatically active domains of PAM, only PHM and not PAL immunoreactivity was found at all stages studied. The early appearance of PAM in developing mouse lung, as well as its presence in a variety of tissues, probably indicates a complex role of this enzyme in pulmonary development and function.

Dynamin is a 100-kD GTPase, which is required for clathrin-mediated endocytosis. Recent studies have revealed that dynamin is closely involved in clathrin-coated vesicle formation. In this study we investigated the ultrastructure of endocytotic structures accumulated in HeLa cells that were transformed with a temperature-sensitive (ts) mutant of dynamin to clarify which step was blocked in dyn^ts cells. Endocytosis of transferrin receptors was restricted at the level of surface-connected membrane structures. Tubular and vesicular membrane invaginations were accumulated in the cells' peripheral regions, suggesting that the endocytosis was blocked just before the pinching-off steps in coated vesicle formation. The “collared” tubes, which were reported to be localized in nerve terminals in shibire^ts1 flies and GTPγS-treated synaptosomes, were not observed in the dyn^ts cells even at nonpermissive temperature. The distribution pattern of dynamin in deeply invaginated coated pits in dyn^ts cells was similar to that in dyn^wt cells but not to that in dyn^K44A cells, which are other endocytosis-defective mutant cells. These morphological data suggest that dyn^ts blocked the pinching-off steps in clathrin-coated vesicle formation, which may be caused by a different mechanism from that of dyn^K44A cells.

Although cardiac effects of growth hormone (GH) and insulin-like growth factor (IGF)-I have been reported in experimental models of heart failure and in human dilated cardiomyopathy, the IGF system has not been comprehensively assessed in the failing heart. We therefore localized the IGF system in the left ventricle during congestive heart failure after myocardial infarction (Ml) in the rat. The left anterior descending coronary artery was ligated in adult female Sprague-Dawley rats and hearts were examined after 6 months when congestive heart failure had developed. In situ hybridization histochemistry was used to localize mRNA for the components of the IGF system in the left ventricle of sham and congestive heart failure animals. We were able to detect changes in the spatial distribution of mRNA for IGF-I and IGF binding proteins 3, 4, 5, and 6 in the left ventricle during congestive heart failure after Ml. IGF-I and the binding proteins were predominantly increased in the infarct/peri-infarct area of the left ventricle. Other components of the IGF system were indistinguishable from the low to undetectable levels in sham-operated rats. These results demonstrate that the IGF system is altered in the failing heart and suggest that the IGF system plays an important role in the response of the heart to Ml and consequent failure.

Destruction of neurons through the genetically directed process of programmed cell death (PCD) is an area of intense interest because this is the underlying mechanism in a variety of developmental and neurodegenerative diseases. The ability to identify and track viable neurons subjected to PCD could be invaluable in development of strategies to prevent or reverse the downstream mechanisms of neuronal PCD. We have developed a novel assay for PCD in viable, adherent cells using annexin V labeling. Annexin V binds to the highly negatively charged plasma membrane phosphatidylserine residues that undergo membrane translocation during PCD. Current annexin V techniques are almost exclusively restricted to flow cytometric analysis. Our unique technique permits repeated examination of individual viable neurons without altering their survival. Correlation with electron microscopy and dye exclusion assays demonstrate both sensitivity and specificity for our method to detect PCD. To our knowledge, this is the first account of a technique that positively identifies PCD in viable, adherent cells.

We used a specific monoclonal antibody to human hepatocyte growth factor activator inhibitor type 1 (HAI-1) in immunohistochemical procedures to determine the distribution and localization of HAI-1 in human tissues. In normal adult tissues, HAI-1 was predominantly expressed in the simple columnar epithelium of the ducts, tubules, and mucosal surface of various organs. In all cases, HAI-1 was localized predominantly on the cellular lateral (or basolateral) surface. By contrast, hepatocytes, acinar cells, endocrine cells, stromal mesenchymal cells, and inflammatory cells were hardly stainable with the antibody, and stratified squamous epithelium showed only faint immunoreactivity on the surface of cells of the basal layer. In the gastrointestinal tract, the surface epithelium was strongly stained. RNA blot analysis confirmed the presence of specific mRNA transcript in the gastrointestinal mucosa, and in situ hybridization revealed that HAI-1 mRNA showed a similar cellular distribution pattern. Although HAI-1 was not expressed in normal hepatocytes, strong immunoreactivity was observed on the epithelium of pseudo-bile ducts and on the surface of scattered hepatocytes in fulminant hepatitis. The enhanced expression was also noted in regenerating tubule epithelial cells of the kidney after infarction. We conclude that HAI-1 is preferentially expressed in the simple columnar epithelium of the mucosal surface and duct, that the predominant localization of HAI-1 is the cell surface, and that the expression of HAI-1 can be modulated by tissue injury and regeneration.

Integration of vascular endothelial function relies on multiple signaling mechanisms, including direct cell-cell communication through gap junctions. Gap junction proteins expressed in the endothelium include connexin37, connexin40, and connexin43. To investigate whether individual endothelial cells in vivo express all three connexin types and, if so, whether multiple connexins are assembled into the same gap junction plaque, we used affinity-purified connexin-specific antibodies raised in three different species to permit multiple-label immunoconfocal and immunoelectron microscopy in the rat main pulmonary artery. Immunoconfocal microscopy showed a high incidence of co-localization between connexin43 and connexin40, but lower incidences of co-localization between connexin37 and connexin40 or connexin43. Immunoelectron microscopy revealed that 83% of gap junction profiles contained all three connexins, with the proportion of connexin40 labeling being significantly higher than that of connexin37 or connexin43. The presence of three different connexin types of distinct properties in vitro provides potential for complex regulation and functional differentiation of endothelial intercellular communication properties in vivo.

We applied in situ hybridization and the TUNEL technique to free-floating (vibratomed) sections of embryonic and postnatal mouse CNS. Full-length cDNAs specific for oligodendrocyte- or astrocyte-specific genes were labeled with digoxigenin using the random primer method. With paraformaldehyde-fixed sections, the nonradioactive in situ hybridization method provides detection of individual, very small glial progenitor cells in embryonic development. Small, isolated cells expressing oligodendrocyte specific messages can be detected in the neuroepithelium at embryonic and postnatal stages. The technique can be completed within 3 days and is as sensitive as the radioactive method. Likewise, the TUNEL method using DAB as the chromogen on free-floating sections provides excellent resolution. These DAB-stained sections can be embedded in plastic and thin-sectioned to visualize the ultrastructure of apoptotic cells. Both in situ hybridization and TUNEL methods can be applied to the same section, the tissue embedded in plastic, and semithin sections cut. The high resolution obtained with this combined procedure makes it possible to determine whether brain cells expressing glia-specific messages are undergoing apoptosis.

Decalcification is routinely performed for histological studies of bone-containing tissue. Although DNA in situ hybridization (ISH) and comparative genomic hybridization (CGH) have been successfully employed on archival material, little has been reported on the use of these techniques on archival decalcified bony material. In this study we compared the effects of two commonly used decalcifiers, i.e., one proprietary, acid-based agent (RDO) and one chelating agent (EDTA), in relation to subsequent DNA ISH and CGH to bony tissues (two normal vertebrae, six prostate tumor bone metastases with one sample decalcified by both EDTA and RDO). We found that RDO-decalcified tissue was not suited for DNA ISH in tissue sections with centromere-specific probes, whereas we were able to adequately determine the chromosomal status of EDTA-decalcified material of both control and tumor material. Gel electrophoresis revealed that no DNA could be successfully retrieved from RDO-treated material. Moreover, in contrast to RDO-decalcified tumor material, we detected several chromosomal imbalances in the EDTA-decalcified tumor tissue by CGH analysis. Furthermore, it was possible to determine the DNA ploidy status of EDTA-but not of RDO-decalcified material by DNA flow cytometry. Decalcification of bony samples by EDTA is highly recommended for application in DNA ISH and CGH techniques.

We describe an in situ DNA nick end-labeling method that can be performed at the electron microscopic level and can also be combined with immunoelectron microscopy. As the materials, we used skin tissues from normal skin and from Bowen's disease that had been cryofixed, freeze-substituted, and embedded in Lowicryl K11M resin. Ultrathin sections were cut and incubated with a reaction buffer containing digoxigenindUTP and terminal deoxynucleotidyl transferase. Digoxigenin nucleotides were labeled with anti-digoxigenin antibodies conjugated with colloidal gold. Specific signals were detected in the condensed chromatin of differentiated epidermal cells and hair follicles in normal skin and of dyskeratotic cells in Bowen's disease. The labeling density over chromosomal areas of apoptotic cells was significantly higher than that over chromosomal areas of mitotic cells or cytoplasmic areas. Ultrastructure was well preserved and double staining with an anti-keratin antibody was also successfully performed. This simple method has a wide range of applications to identify the nature of apoptotic cells and explore the mechanisms of apoptosis.