Histochemical demonstration of zinc in rat epididymis using a sulphide-silver method

Summary We studied the histochemical distribution of zinc in rat epididymis using a sulphide-silver method. In the supranuclear cytoplasm of the principal cells that line the epididymis of rats, varying amounts of sulphide-silver-reactive zinc were visualized. In adult mating rats, significant amounts of zinc were found in the proximal portion of the epididymis, whereas in non-mating, mature and immature young rats, this heavy metal was most prominent in the distal portion of this organ. In all of the rats studied, zinc was sparsely distributed in the intermediate portion of the epididymis. From these results, it can be assumed that the zinc present in the epithelial lining of rat epididymis plays an important role in the maturation of spermatozoa. The present results represent a useful contribution to our understanding of the functional morphology of rat epididymis.Dedicated to Professor Dr. T.H.Schiebler on the occasion of his 65th birthday     

Histochemical demonstration of zinc in rat epididymis using a sulphide-silver method

We studied the histochemical distribution of zinc in rat epididymis using a sulphide-silver method. In the supranuclear cytoplasm of the principal cells that line the epididymis of rats, varying amounts of sulphide-silver-reactive zinc were visualized. In adult mating rats, significant amounts of zinc were found in the proximal portion of the epididymis, whereas in non-mating, mature and immature young rats, this heavy metal was most prominent in the distal portion of this organ. In all of the rats studied, zinc was sparsely distributed in the intermediate portion of the epididymis. From these results, it can be assumed that the zinc present in the epithelial lining of rat epididymis plays an important role in the maturation of spermatozoa. The present results represent a useful contribution to our understanding of the functional morphology of rat epididymis.     

Removal of fibroblastoid cells from primary monolayer cultures of rat neonatal endocrine pancreas by sodium ethylmercurithiosalicylate

Anti-cytochrome b₅ immunoglobulin (AIg) from a rabbit was used to establish the role of cytochrome b₅ in the transfer of electrons from NADH or NADPH to the hepatic microsomal mono-oxidase system of the rat. AIg inhibited ethylmorphine (EM) N-demethylase when both NADH and NADPH were present, but had little effect when NADPH was the only source of electrons. Inhibition was reversed when AIg was preincubated with pure cytochrome b₅. Specificity of AIg was shown by its inhibitory effect on NADH cytochrome c reductase activity; it was without effect on NADPH-cytochrome P-450 reductase or aniline hydroxylase activities. It is concluded that the second electron required for EM N-demethylation can be donated by NADH via cytochrome b₅.     

Histochemical demonstration of acid mucopolysaccharide production by tissue cultured epithelial-like cells

Summary The production of mucopolysaccharides by human epithelial-like cells in vitro has been studied using histochemical techniques. Cells of the human amnion F/L line were grown for 12–14 days in Leighton tubes containing cover slips which were fixed and stained by the Hale, PAS, toluidine blue and alcian blue stains and techniques. Control slides were treated with active and heat inactivated solutions of testicular and bacterial hyaluronidase and chondroitinase prior to staining. Results indicated that material stainable by all the techniques employed was elaborated by the amnion cells and this was partially removable by various combinations of polysaccharases. It is concluded that epithelial-like cells can synthesize and elaborate the ground substance components, hyaluronic acid, chondroitin sulphates and unidentified polysaccharide complexes, in much the same manner as mesenchymal cells and thus contribute to the maintenance of epithelial integrity by de novo production of intercellular substances.Supported by USPHS Grants No. 991 and DE-01681, T01-DE00108 National Institute of Dental Research, National Institutes of Health and the National Health and Medical Research Council, Australia.     

Histochemical demonstration of carbohydrates by a convenient sulphation technique

Summary A simple and convenient procedure has been devised for the staining of carbohydrate derivatives in tissue sections. Dewaxed sections are sulphated either by immersion in a 1:1 acetic-sulphuric acid mixture for 10–15 minutes or by exposure to sulphuryl chloride vapour for 15–20 minutes. The slides are then stained in a 0.05% solution of methylene blue buffered to aph of 2.4. Highly selective staining of tissue carbohydrates is obtained. The acetic-sulphuric mixture rapidly hydrolyses glycogen which is therefore lost, but the sulphuryl chloride technique produces intense staining of glycogen. The various steps in the technique are discussed, with particular emphasis on the reasons for the final choice of conditions. These sulphation techniques have several advantages over the PAS procedure: the overall picture obtained is clearer; definition at high magnifications is often better; and the versatility of the technique can be widened by varying the precise conditions of sulphation and staining to suit the particular problem being studied.

---

Zusammenfassung Es wird eine einfache und handliche Methode zur Darstellung von Kohlenhydratderivaten in Gewebsschnitten beschrieben. Entparaffinierte Schnitte werden 10–15 min in einem 1:1 Essigsäure-Schwefelsäure-Gemisch bzw. durch Behandlung mit Sulfurylchloriddampf während 15–20 min sulfatiert und anschließend mit einer 0,5%igen Methylenblaulösung (gepuffert aufph 2,4) gefärbt. Man erhält eine stark selektive Färbung der Gewebskohlenhydrate. Das Essigsäure-Schwefelsäure-Gemisch hydrolysiert und entfernt das Glykogen sehr rasch. Demgegenüber ist nach Sulfurylchloridbehandlung eine starke Anfärbung von Glykogen möglich. Die einzelnen Schritte der Technik werden diskutiert, besonders die Gründe, die uns bei der endgültigen Wahl der Methodik geleitet haben. Die Methode bietet verschiedene Vorteile gegenüber der PAS-Reaktion: Das Übersichtsbild ist klarer, die Schärfe ist bei starken Vergrößerungen oft besser; die Plastizität der Methode ermöglicht es, Sulfatierung und Färbung den Bedürfnissen verschiedener Probleme anzupassen.

---

---

With 5 Figures in the Text     

Immunocytochemical demonstration of ornithine decarboxylase in the rat exocrine pancreas using the protein A-gold technique

Previous studies from our laboratory have shown that caerulein, a cholecystokinin analog, can induce pancreatic growth. Because ornithine decarboxylase (ODC) could be involved in this process, it is of interest to localize and estimate ODC immunoreactivity in rat pancreatic acinar cells from control and caerulein-treated animals. This was carried out with the protein A-gold immunocytochemical technique. Rats received either saline (control) or caerulein at a dose of 1 microgram X kg-1 and were sacrificed 8 h after the first injection (control and caerulein group), 4 h after the second caerulein injection (second caerulein group), and 8 h after the third caerulein injection (third caerulein group). ODC immunoreactivity was revealed using a specific antibody. ODC was localized specifically in nuclei and rough endoplasmic reticulum (RER) of the pancreatic acinar cells and the number of gold particles was increased in both of these organelles by caerulein. Peak ODC immunoreactivity was observed in nuclei 4 h after the second caerulein injection, whereas it occurred 8 h after the third peptide injection in the RER. These studies are the first to demonstrate ODC localization in pancreatic acinar cells and show that the enzyme can be induced early upon growth stimulation of the organ by a cholecystokinin analog.     

Rearrangements of desmosomal and cytoskeletal proteins during the transition from epithelial to fibroblastoid organization in cultured rat bladder carcinoma cells

Changes of cell morphology and the state of differentiation are known to play important roles in embryogenesis as well as in carcinogenesis. Examples of particularly profound changes are the conversions of epithelial to mesenchymal cells; i.e., the dissociation of some or all polygonal, polar epithelial cells and their transformation into elongate, fibroblastoid cells of high motility. As an in vitro model system for such changes in cell morphology, we have used cell cultures of the rat bladder carcinoma-derived cell line NBT-II which, on exposure to inducing medium containing a commercial serum substitute (Ultroser G), show an extensive change in their organization (epithelial-mesenchymal transition): the junctions between the epithelial cells are split, the epithelial cell organization is lost, and the resulting individual cells become motile and assume a spindle- like fibroblastoid appearance. Using immunofluorescence microscopy and biochemical protein characterization techniques, we show that this change is accompanied by a redistribution of desmosomal plaque proteins (desmoplakins, desmoglein, plakoglobin) and by a reorganization of the cytokeratin and the actin-fodrin filament systems. Moreover, intermediate-sized filaments of the vimentin type are formed in the fibroblastoid cells. We demonstrate that the modulation of desmosomal proteins, specifically an increase in soluble desmoplakins, is a relatively early event in cell dissociation and in epithelial- mesenchymal transition. In this process, a latent period of 5 h upon addition of inducing medium precedes the removal of these desmosomal components from the plasma membrane. The transition, which is reversible, is dependent on continued protein synthesis and phosphorylation but not on the presence of the inducing medium beyond the initial 2-h period. We discuss the value of this experimental system as a physiologically relevant approach for studying the regulation of the assembly and disassembly of desmosomes and other intercellular adhesion structures, and as a model of the conversion of cells from one state of differentiation into another.     

Histochemical demonstration of apoptotic cells in the chicken embryo using annexin V

This study describes the use of biotinylated annexin V for the histochemical detection of apoptotic cells in cultured chicken embryos during gastrulation. This method is based on the Ca2+-dependent binding of annexin V to phosphatidylserine, a negatively charged phospholipid, located at the inner leaflet of the cell membrane in living cells. However, in the early stages of apoptosis, phosphatidylserine is translocated to the outer layer of the cell membrane and can then be recognized by annexin V. Applying this method in cultured chicken embryos during gastrulation, we obtained labelling of apoptotic cells in the three germ layers. In the epiblast and mesoblast, labelling was predominantly present in the region lateral to the primitive streak. At the level of the germinal crescent, labelled cells were also found in the epiblast. Labelled cells in the deep layer, which is a heterogeneous tissue layer composed of endophyll, sickle endoblast and definitive endobl ast, were rather scarce. The distribution of cells, as observed in this study after labelling with annexin V in light microscopy and confocal laser scanning microscopy, is consistent with distributions reported by other authors using other approaches and with our previous observations made with the TUNEL technique and by electron microscopy after fixation in a tannic acid-based fixative. The main advantages of this method over other more sophisticated methods is its easiness and rapidity of execution and the fact that both early and late stages of apoptosis are detected. © 1998 Chapman & Hall     

The histochemical demonstration of fructose diphosphate aldolase activity using a semipermeable membrane technique

Summary In this communication an enzyme histochemical multistep technique for the demonstration of class 1 fructose-1,6-diphosphate aldolase in heart and skeletal muscle sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the enzyme into the medium during incubation. In the histochemical system the enzyme cleaves the substrate D-fructose-1,6-diphosphate to dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triose phosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase and the electrons are transported concomitantly via NAD⁺, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.     

Histochemical study of apoptotic epithelial cells depending on testosterone in primary cultured rat prostatic tissues

To clarify whether apoptosis can be induced in cultured rat prostatic epithelial cells, they were investigated at various time points, depending on different concentrations of testosterone. Ventral lobes of rat prostates were cultured as small pieces of tissues up to 14 days. They were examined by anti-Fas antibody immunostaining and also compared to findings revealed by in situ end-labelling (ISEL) technique. To clarify apoptotic nuclei at high resolution, the quick-freezing and deep-etching (QF-DE) method was also used, as reported before. The localization and appearance of Fas-positive cells were detected more widely and earlier than those of ISEL-positive cells, but both label-positive localizations were closely related to each other. In addition, they were detected more often in epithelial cells cultured with low testosterone concentrations. By the QF-DE method, chromatin fibers were found to be broken in spotty parts of apoptotic nuclei. We could control the concentration of testosterone in culture medium and detect the appearance of Fas antigen in cultured prostatic epithelial cells, followed by apoptotic changes. So, Fas and Fas-ligand system is one candidate for apoptosis in the prostate glands, depending on removal of hormonal testosterone.     

Histochemical demonstration of copper in normal rat brain and spinal cord

Summary The high sensitivity of the magnesium-dithizonate silver-dithizonate (MDSD) staining procedure makes this method very suitable for the histochemical localization of copper in different regions of the central nervous system of adult rats. In the telencephalon (bulbus olfactorius, nucleus caudatus-putamen, septum pellucidum and are dentata), diencephalon (nucleus habenulae medialis, nuclei of the hypothalamus in the vicinity of the third ventricle, and corpus mamillare), mesencephalon (substantia nigra), cerebellum (mainly in the nodulus), pons (locus coeruleus, nucleus vestibularis), medulla oblongata (nucleus tractus solitarii) and spinal cord, the glial cells exhibit specific copper staining. The glial cells of some circumventricular organs (e.g. the subfornical organ) are also stained using the MDSD method. The significant staining observed in whitematter glial cells (e.g. in the corpus callosum, cerebellum and spinal cord) further indicates the very high sensitivity of this method. In glial cells of the same regions, the presence of copper can likewise be demonstrated using the modified sulphide silver method. On the basis of the present histochemical results, it is suggested that copper may play an important role in the normal physiological functioning of glial cells and also, via glia-neuron interactions, in neuronal processes.