Histochemical localization of zinc ions in the epididymis of the rat

Summary In the present study, the autometallograpic zinc sulphide technique, an improved version of the original Timm sulphide-silver method, was used. This technique reveals a particular pool of ionic zinc that is chelatable by diethyldithiocarbamate. At the light microscopical level, no reaction for zinc was found in tissues of young prepubertal rats. In adult mating and non-mating rats low zinc staining was found in the head and intermediate epididymis whereas the tail of the epididymis demonstrated high levels of zinc ions. Sections from the epididymal tail revealed a compartmentalization, based on pronounced differences in staining intensity along the epididymal ducts. At higher magnification zinc ions were found in the apical part of the principal cell and in the lumen. At the ultrastructural level autometallographic grains were located in vesicles and in lysosome-like structures of the apical parts of the principal cells. The luminal grains were found either associated with sperm cells, with the surface of the large microvilli (stereocilia), or free in the seminal fluid. The variation in content of zinc ions in the epididymal epithelium and lumen suggests that zinc ions are secreted into the lumen from the epididymal tail and may somehow be involved in maturation of the sperm cells.     

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.     

In situ hybridization: Alkaline phosphatase visualization of biotinylated probes in cryostat and paraffin sections

Summary Alkaline phosphatase immunochemical systems were evaluated for use in the demonstration ofin situ hybridized biotin-labelled probes in frozen and fixed sections of tonsil. Three probes were used: total genomic DNA, pHY2.1, a human repetitive sequence which hybridizes to a 2.12 KB sequence on the Y chromosome (2000 repeats) and a 2.0 KB sequence on the autosomes (100–200 repeats), and human papilloma virus type II. Indirect, three- and five-stage detection methods were compared on cryostat sections. The indirect method involved the application of a streptavidin, biotinylated alkaline phosphatase sequence. The three-stage procedure comprised a mouse monoclonal anti-biotin, rabbit anti-(mouse immunoglobulin), mouse APAAP system. In the five-stage method the indirect and three-stage reagents were sequentially applied. Alkaline phosphatase was demonstrated using a Fast Red naphthol-capture method.The total genomic DNA probe was used initially to investigate hybridization conditions including the optimum temperature of denaturation, which was found to be higher than previously reported. The five-stage detection method gave the most sensitive results for the Y sequence probe, with intense demonstration of the Y body in male nuclei and autosomal sequences in female nuclei. This method was then applied to fixed tissue sections and gave Y body signals on Bouin's and Carnoy's fixed tissue. On the other hand tissue fixed using formalin-based solutions required proteolytic digestion as a pretreatment to hybridization for a Y body signal. The application of this methodology to viral diagnosis in routine fixed anogenital tissue and cytological preparations was also demonstrated.     

Influence of fixatives on the immunoreactivity of paraffin sections

Summary The unlabelled antibody peroxidase-antiperoxidase (PAP) technique was used to demonstrate IgA in paraffin sections of human tonsil fixed in eight commonly known fixatives. In all but one of the fixatives, treatment with trypsin prior to immunostaining resulted either in digestion of sections, or no change in positive staining. In tissues fixed in neutral buffered formalin-saline, positive staining was absent unless the sections had been treated with trypsin, and it has been shown that digestion requirements of different areas throughout a section may vary according to the effectiveness of fixation.     

The potential of the immunogold-silver staining method for paraffin sections

Summary After perfusion fixation of the rat kidney with glutaraldehyde, and postfixation of the renal cortex with osmium-low ferrocyanide (40 mM OsO₄+6 mM K₄Fe(CN)₆ in 0.135 M phosphate buffer, pH 8.0), secondary lysosomes of proximal tubule cells carry acoat of electron dense material on the inner surface of the lysosomal membrane. This coat separates matrix and membrane of lysosomes, and corresponds in location and width to the electron translucent halo of conventionally processed lysosomes in TEM. The material which forms the coat, is stained by phosphotungstic acid at pH 0.3, and by periodic acid — thiocarbohydrazide — silver proteinate more intensively than the cell surface coat of the same cell; it contains a high concentration of hydroxyl,vicinal-glycol and α-aminoalcohol groups. Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Structural organization of rat hepatocyte chromatin as visualized in thin frozen sections selectively stained for DNA

We studied the structure of rat hepatocyte chromatin in situ using thin frozen sections selectively stained for DNA after aldehyde fixation. Our results indicate that intranucleolar chromatin is arranged into three different organization levels, confirming the observations on Epon-embedded chromatin. These are: completely extended DNA filaments, with a thickness of approximately 3 nm, clustered in loose, roundish agglomerates, very long fibers with a thickness ranging from 15 to 35 nm and compact chromatin clumps. Both the fibers and the chromatin clumps frequently appeared to be composed of nucleosome-like particles. In the extranucleolar chromatin, agglomerates of extended DNA filaments and long fibers were never visualized. In contrast to data from Epon-embedded chromatin, we noticed that in frozen sections neither the nucleolar nor the extranucleolar compact chromatin appear to be organized into discrete, 20 to 30 nm superordered fibers.     

ProteomIQ blue, a potent post-stain for the visualization and subsequent mass spectrometry based identification of fluorescent stained proteins on 2D-gels

Manual spot excision for protein identification from fluorescent stained two-dimensional (2-D) gels is hard to accomplish. Here, we explore the use of ProteomIQ Blue as a post-stain method for the visualization of fluorescent stained/labeled proteins. We show that ProteomIQ Blue post-staining is almost as sensitive as staining with SYPRO Ruby or cyanine dyes alone. More than 90% of the protein spots that are stained with the fluorescent stains are still detectable with ProteomIQ Blue. In protein identification by mass spectrometry, ProteomIQ Blue post-stained spots provide high sensitivity and high protein sequence coverage of the peptide mass maps in both MALDI-TOF-MS and ESI-MS/MS analyses. In conclusion, post-staining of fluorescent stained gels with ProteomIQ Blue provides a facile and a powerful method to achieve quantitative protein analysis as well as protein identification in the same semianalytical gel without requiring sophisticated/expensive robotic equipment.     

Identification of tissue histiocytes on paraffin sections by a new monoclonal antibody

A mouse monoclonal antibody (MAC 387) with specificity for monocytes and tissue histiocytes was produced by immunization of a BALB/c mouse with peripheral blood monocyte components derived by affinity chromatography of detergent-solubilized monocyte material on Sepharose 4B coupled to rabbit anti-monocyte antibodies. MAC 387 strongly stained the cytoplasm of cells of the monocyte/macrophage series on paraffin sections after controlled trypsinization of sections. The antibody showed broad reactivity for a variety of tissue histiocytes, including infiltrating and reactive histiocytes, alveolar macrophages, Kupffer cells, follicle-center macrophages, splenic red pulp macrophages, tumor-infiltrating macrophages, sinus histiocytes, epithelioid giant cells (variably), and cases of histiocytosis X and dermatopathic lymphadenopathy. Molecular weight data obtained by Western blotting, immunoprecipitation, and immunoaffinity-purification revealed that the antigen was present in different forms in the monocyte and granulocyte. In the granulocyte, free alpha (Mr 12 KD) and beta (Mr 14 KD) chains expressing the MAC 387 epitope were found together with associations of one alpha and one beta chain linked by disulfide bonds to yield a heterodimer of Mr 26 KD. In the monocyte, free alpha and beta chains are not found, but instead the heterodimer and associations of two (Mr 56 KD) and four (Mr 112 KD) heterodimers are disulfide-linked together. This new monoclonal reagent should have particular value for identification of tissue histiocytes in routine paraffin sections and particularly for demonstration of histiocytes in malignant lymphomas.     

Buffered Romanowsky-Giemsa method for formalin fixed,paraffin embedded sections: taming a traditional stain

Romanowsky-Giemsa (RG) stains were devised during the 19th century for identifying plasmodia parasites in blood smears. Later, RG stains became standard procedures for hematology and cytology. Numerous attempts have been made to apply RG staining to formalin-fixed paraffin-embedded tissue sections, with varied success. Most published work on this topic described RG staining methods in which sections were overstained, then subjected to acid differentiation; unfortunately, the differentiation step often caused inconsistent staining outcomes. If staining is performed under optimal conditions with control of dye concentration, pH, solution temperature and staining time, no differentiation is required. We used RG and 0.002 M buffer, pH 42, for staining and washing sections. All steps were performed at room temperature. After staining and air drying, sections were washed in 96?100% ethanol to remove extraneous stain. Finally, sections were washed in xylene and mounted using DPX. Staining results were similar to routine hemalum and eosin (H &; E) staining. Nuclei were blue; intensity depended largely on chromatin density. RNA-rich sites were purple. Collagen fibers, keratin, muscle cells, erythrocytes and white matter of the central nervous system were stained pinkish and reddish hues. Cartilage matrix, mast cell granules and areas of myxomatous degeneration were purple. Sulfate-rich mucins were stained pale blue, while those lacking sulfate groups were unstained. Deposits of hemosiderin, lipofuscin and melanin were greenish, and calcium deposits were blue. Helicobacter pylori bacteria were violet to purple. The advantages of the method are its close similarity to H &; E staining and technical simplicity. Hemosiderin, H. pylori, mast cell granules, melanin and specific granules of different hematopoietic cells, which are invisible or barely distinguishable by H &; E staining, are visualized. Other advantages over previous RG stains include shorter staining time and avoidance of acetone.     

Genetic immunization: a new monoclonal antibody for the detection of BCL-6 protein in paraffin sections.

Genetic immunization can be combined with hybridoma technology to generate high-affinity monoclonal antibodies (MAbs). A new anti-BCL-6 MAb (GI191E/A8) was produced by cloning full-length BCL-6 cDNA into a eukaryotic vector and delivering this into mouse epidermis using a helium gene gun. A comparative study was made of the specificity and the effects of formalin fixation on immunohistochemistry quality of GI191E/A8 and two other anti-BCL-6 MAbs. To evaluate its possible application to differential diagnosis of lymphomas, two tissue microarrays (89 diffuse large B-cell lymphomas and 24 B-cell chronic lymphocytic leukemia cases) were stained with GI191E/A8 and another anti-BCL-6 MAb produced by conventional means. Using GI191E/A8, the detection of BCL-6 protein was significantly increased, and its specificity was independent of formalin-fixation time. Using automatic quantified analysis, the correlation between the two anti-BCL-6 MAbs tested was identical in cases with overexpression or absence of BCL-6. In cases with intermediate BCL-6 protein expression, detection with GI191E/A8 was more sensitive. A significant association of higher BCL-6 expression and longer median overall survival times in diffuse large B-cell lymphomas was found. Using conventionally produced MAbs in the same patient group, the association was not significant.     

In situ hybridization: use of 35S-labeled probes on paraffin tissue sections

The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with (35)S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided.     

Apparatus for measuring rat body volume: a methodological proposition.

We propose a communicating-vessels system to measure body volume in live rats through water level detection by hydrostatic weighing. The reproducibility, accuracy, linearity, and reliability of this apparatus were evaluated in two tests using previously weighed water or six aluminum cylinders of known volume after proper system calibration. The applicability of this apparatus to measurement of live animals (Wistar rats) was tested in a transversal experiment with five rats, anesthetized and nonanesthetized. We took 18 measurements of the volume under each condition (anesthetized and nonanesthetized), totaling 90 measurements. The addition of water volumes (50-700 ml) produced a regression equation with a slope of 1.0006 +/- 0.0017, intercept of 0.75 +/- 0.81 (R(2) = 0.99999, standard error of estimate = 0.58 ml), and bias of approximately 1 ml. The differences between cylinders of known volumes and volumes calculated by the system were <0.4 ml. Mean volume errors were 0.01-0.07%. Among the live models, the difference between the volumes obtained for anesthetized and nonanesthetized rats was 0.31 +/- 2.34 (SD) ml (n = 90). These data showed that animal movement does not interfere with the volume measured by the proposed apparatus, and neither anesthesia nor fur shaving is needed for this procedure. Nevertheless, some effort should be taken to eliminate air bubbles trapped in the apparatus or the fur. The proposed apparatus for measuring rat body volume is inexpensive and may be useful for a range of scientific purposes.     

苏木精-伊红染色制作骨骼肌组织教学切片的方法优化

目的优化骨骼肌教学切片的制作方法,以期提高制片效率与切片质量。方法石蜡包埋、苏木精-伊红染色,比较不同物种之间骨骼肌组织以及不用厚度的骨骼肌组织切片横纹结构的差别。结果人与狗的骨骼肌肌原纤维及横纹结构典型,厚度4~5μm 的切片中肌原纤维及横纹结构较其他厚度更易分辨。结论狗的骨骼肌更适合替代人的骨骼肌用于组织教学切片的制作,4~5μm 的切片厚度易于显示横纹结构。