Basic and applied histochemistry最新文献

Guanylate cyclase activity was demonstrated in the rat hippocampus at the electron microscopy level by a cerium precipitation technique. Enzymatic activity is localized in the granular cells of the dentate gyrus and in other, medium-sized neurons. The reaction product was found on the cytoplasmic membrane, in the cytoplasm, on the surface of endoplasmic reticulum and in postsynaptic densities. Glial cell processes and small dendritic processes were also stained. Besides the neurons, glial cells contained strong enzymatic activity, especially the perivascular glia and protoplasmatic glial cells neighbouring to neurons. Only little activity was detected in pyramidal cells and no activity was found in the endothelium cells of capillaries.

The merits of low molecular weight reagents (mini-stains) for localising tissue components, are compared with those of high molecular weight (macro-stains), with respect to stoichiometry, ease of tissue penetration, and particularly specificity. Macro-stains, which are based mainly on antibodies, are likely to be more specific, but this will not always be the case. The concept of specificity in the design of mini-stains is examined, and its application to the delivery of e.g. Cupromeronic blue to particular substrates in the "critical electrolyte concentration" system, is discussed. The much greater resolving power of mini-stains in electron histochemistry has permitted the elucidation of four specific binding sites for proteoglycans along the collagen fibrils, validating the 'one proteoglycan: one binding site' hypothesis.

We describe the immunohistological detection of in vivo interferon-beta (IFN-beta)-induced H-2 antigens in mouse brain tissues. Two injections of recombinant mouse interferon-beta (rIFN-beta) were given into the lateral ventricles. Following perfusion with periodate-lysine-paraformaldehyde (PLP) solution, microsliced brain sections were incubated with anti H-2 monoclonal antibody and these subjected to a streptavidin biotin immunohistochemistry technique. Expression of H-2 antigens was demonstrated on neurons, neuroglias and vascular endothelial cells in the hippocampus, cerebral cortex, corpus callosum and other regions of the brain as well as on ependymocytes in the choroid plexus. H-2 antigens were also diffusely distributed in the cytoplasm and axons.

The distribution of laminin, type IV collagen and fibronectin was studied by immunofluorescence in rat, pig and cow ovarian follicles. The results obtained in the three species investigated were similar. In all the follicles, laminin and type IV collagen were identically localized in the basal lamina (BL) separating the granulosa and the theca layers. In addition, these two proteins were also distributed in the wall of blood vessels of the thecae and ovarian stroma. The staining showed that the BL of primordial and growing follicles was regular and continuous, but underwent striking modifications during ovulation and atresia. In fact, in preovulatory follicles the BL appeared thinner and discontinuous, whereas it was much thickened and ruptured in atretic follicles. Fibronectin was localized mainly in inner granulosa cells of small and medium-sized growing follicles, and as a broad and irregular layer around the cavity of the degenerated follicles. The results show that each stage of follicular growth and involution is associated with a precise and peculiar pattern of distribution of laminin, type IV collagen and fibronectin. The possibility that these proteins play a role in the local control of ovarian follicular dynamics is advanced.

The Harderian gland in Rana esculenta has been studied during the annual cycle at the histological, histochemical and ultrastructural levels. The Harderian gland has an acinar structure and is the only orbital gland in anuran amphibia. It develops at the medial corner of the orbit from the conjunctival epithelium at the premetamorphic stage. In the adult the glandular secretion reaches a maximum during the months of July and August, drops in September and resumes slowly from October onwards. The secretion is seromucoid and the secretory granules are released into the acinar lumen, mainly by exocytosis. Porphyrins were not detected. No sexual dimorphism was observed in the glandular cells. The resumption of secretory activity in October and the enhancement of secretion in May are marked by the appearance of "blue nuclei" (Mallory stain) in a relatively high percentage of glandular cells. This unusual blue colour, using the Mallory stain (by which nuclei stain red), disappears after digestion of paraffin sections with RNAase, but not with DNAase and trypsin. The blue staining may, therefore, indicate an increased amount of nuclear RNA. The Harderian gland in the frog most probably serves to lubricate and moisten the eye in the absence of the lacrimal gland. However, the gland may also represent an immunoactive organ owing to the presence of numerous mast cells and plasma cells in the interacinar spaces.

We have performed immunofluorescent and fluorescent in situ hybridization studies in order to better clarify the integration of SV40 DNA in human fibroblast cell lines. Most of the cells were T-antigen positive by immunocytochemical studies, while in all the cells we detected the integrated viral DNA by in situ hybridization. Both techniques are easy and useful to perform but the molecular genetic method gives a more specific signal with the possibility of localizing molecular hybrids in the nucleus and in the cytoplasm of the transformed cells.

Histochemical study by traditional staining methods (AB, PAS, HID) and by the use of five peroxidase-labelled lectins (ConA, WGL, WPL, SBL, PNL) were carried out to characterize glycoconjugates in the secretory cells of the nasal mucosa of the Lacertid lizard Podarcis sicula campestris De Betta. The mucus covering the nasal epithelium is produced by the supporting cells and the Bowman glands in the olfactory area, and by typical goblet cells and, probably, a second type of secretory cell, in the non-sensory area. Neutral glycoconjugates containing N-acetyl-D-glucosamine and terminal N-acetyl-D-galactosamine, D-mannose and D-glucose residues were present in the secretory product of the Bowman glands. L-fucose and D-galactose were absent. In the supporting cells the secretory product consisted mainly of sulfated glycoproteins containing D-galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, D-mannose, D-glucose, but not L-fucose. Glycoconjugates containing terminal sialic acid and penultimate D-galactose were present in typical goblet cells as was N-acetyl-D-glucosamine.

The localization and quantitation of glycosaminoglycans classes (GAGs) were studied in human meningiomas. Meningiomas presented high amounts of these compounds and electrophoretic separation revealed that they were 90% sulphated. The Alcian method and a polyclonal antiserum against chondroitin sulphate were used to localize the different GAGs in tissue sections. Quantitative and qualitative differences and different tissue distributions of GAGs were observed among transitional, syncytial and fibroblastic meningiomas. Syncytial meningiomas presented the lowest amount of GAGs and the immuno- and histochemical studies showed that they were located only in vessels and connectival trabeculae. Transitional meningiomas contained the highest concentration of GAGs; the percentage of the different GAG classes was similar to that observed in the syncytial oncotype indicating a quantitative but not qualitative difference between the two oncotypes. The high amount of GAGs in transitional meningiomas was attribute to the whorls, the structures stained by the histochemical and immunohistochemical techniques. The tumoral parenchyma of these two oncotypes was negative. On the contrary, fibroblastic meningiomas showed a fine meshwork among tumoral cells containing chondroitin sulphate and heparan sulphate. Biochemical data were consistent with the histochemical and immunohistochemical findings revealing a high percentage of chondroitin sulphate and heparan sulphate in fibroblastic meningiomas. This study suggests that the three meningioma types have different abilities to produce extracellular matrix components.

We utilized the fluorescent serotonin analogue 5,7-dihydroxytryptamine (5,7DHT) to visualize basal cells in the frog's taste organ in supravital conditions. In whole mounts of lingual mucosa, specifical and detailed morphological visualization of fluorescent basal cells was obtained in the peripheral and central region of the intact taste organ; similar results were obtained after mechanical dissociation. Preincubation with serotonin prevented any fluorescence in basal cells. Electron microscopy showed good preservation of the ultrastructural morphology of the taste disk after exposure to 5,7DHT. The advantages of the current method as compared with conventional ones are discussed. This simple, reliable procedure will be useful to further define the biology of neuroendocrine cells in taste as well as in other organs.