Journal of Histochemistry & Cytochemistry

Wirt, D P; Grogan, T M; Nagle, R B; Copeland, J G; Richter, L C; Rangel, C S; Schuchardt, M; Fosse, J; Layton, J M

doi: 10.1177/36.1.2961798pmid: 2961798

We have achieved a comprehensive immunotopographic mapping of human thymus by using a large battery of monoclonal antibodies and the methodological refinement of comparative serial tissue section immunohistochemistry, allowing analysis of multiple phenotypes in the same tissue site. Previous immunohistochemical studies of thymus have concentrated on the majority T-cell and epithelial cell populations. Besides demonstrating the complexity of T-cell antigenic expression (e.g., simultaneous cortical expression of Leu 2, Leu 3, CALLA, Tdt, and Leu 6), we delineate surprisingly complex B-cell zones (e.g., septal B-follicles with DRC+C3d+ dendritic cells and zonal maturation of B-cells). Whereas septal B-follicles were found in 25% of cases, medullary B-cells were universally present as a substantial minority component. This expanded immunotopographic knowledge of the complex T-, B-, epithelial, and reticulum cell neighborhoods suggests that the thymus is an organ capable of a broad repertoire of immunological responses, not limited to T-cell development.

Hepler, J R; Toomim, C S; McCarthy, K D; Conti, F; Battaglia, G; Rustioni, A; Petrusz, P

doi: 10.1177/36.1.2891743pmid: 2891743

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.

Angermüller, S; Fahimi, H D

doi: 10.1177/36.1.2891744pmid: 2891744

The reaction product of cerium used for localization of peroxisomal oxidases is highly electron-dense but lacks sufficient contrast at the light microscopic level. We describe two methods for converting the reaction product of cerium to colored compounds visible by light microscopy. The first method is based on 3,3'-diaminobenzidine (DAB) amplification of transition metal compounds, of which cerium is one. Sections of glutaraldehyde-fixed rat liver or kidney are incubated first in media for various oxidases containing CeCl3, followed by treatment with DAB in Na acetate buffer, pH 5.3. To prevent any interference by the peroxidatic activity of catalase, NaN3 or Na pyruvate is added to the DAB amplification medium. Staining with DAB can be further intensified with NiCl2 or CoCl2. The second method is based on the conversion of the cerium reaction product with alkaline lead citrate and the final visualization of the lead compound with ammonium sulfide. These methods allow the evaluation of large sections for peroxisomal oxidases by light microscopy, making close correlation between light and electron microscopy possible.

Hamano, Y; Kodama, H; Yanagisawa, M; Haraguchi, Y; Mori, M; Yokota, S

doi: 10.1177/36.1.3275711pmid: 3275711

We investigated light and electron microscopic localization of ornithine transcarbamylase (OTC) in rat intestinal mucosa. In the immunoblotting assay of OTC-related protein, a single protein band with a molecular weight of about 36,500 is observed in extracts of liver and small intestinal mucosa but is not observed in those of stomach and large intestine. For light microscopy, tissue slices of the digestive system were embedded in Epon and stained by using anti-bovine OTC rabbit IgG and the immunoenzyme technique. For electron microscopy, slices of these and the liver tissues were embedded in Lowicryl K4M and stained by the protein A-gold technique. By light microscopy, the absorptive epithelial cells of duodenum, jejunum, and ileum stained positively for OTC, but stomach, large intestine, rectum, and propria mucosa of small intestine were not stained. Electron microscopy showed that gold particles representing the antigenic sites for OTC were confined to the mitochondrial matrix of hepatocytes and small intestinal epithelial cells. However, the enzyme was detected in mitochondria of neither liver endothelial cells, submucosal cells of small intestine, nor large intestinal epithelial cells. Labeling density of mitochondria in the absorptive epithelial cells of duodenum, jejunum, and ileum was about half of that in liver cells.

Schaumann, L; Galle, P; Thellier, M; Wissocq, J C

doi: 10.1177/36.1.3335768pmid: 3335768

Thanks to the "secondary-ion emission microscope" (CAMECA IMS 300), we have been able to image the distribution of the stable isotopes of nitrogen 14N and 15N in sections of plant roots (spatial resolution better than 1 micron), as well as to estimate the relative concentrations of these isotopes. The plants used (Lupinus spec.) originated from seeds with natural (i.e., 14N) nitrogen and had been fed for a few days with [15N]-nitrate before sampling. We have found in root sections of 6-day-old plants (prepared at 5 mm from the root tip) a clear-cut regionalization of the distribution of 15N between the vascular cylinder and the cortex. The latter contained approximately 5% 15N (of total nitrogen), whereas the relative concentration of the heavy isotope in the vascular cylinder was significantly lower. The observed concentration difference is probably due to the Casparian strip, which is a barrier for the apoplastic diffusion of solutes from the cortex to the vascular cylinder.

Lamers, W H; Janzen, J W; Moorman, A F; Charles, R; Knecht, E; Martínez-Ramón, A; Hernández-Yago, J; Grisolía, S

doi: 10.1177/36.1.3335769pmid: 3335769

In adult rat liver, glutamate dehydrogenase is present in high concentrations around the terminal portal (zone 1) and hepatic (zone 3) veins, whereas its concentration is low in the intermediate zone. Although the size and staining intensity of the periportal glutamate dehydrogenase-positive compartment are less than those of the pericentral compartment, it can expand under appropriate endocrine conditions, leading to a homogeneous distribution. At birth, glutamate dehydrogenase is also homogeneously distributed. Glutamate dehydrogenase disappears from the periportal compartment during the first postnatal week and reappears in that compartment after weaning. These observations indicate an independent regulation of glutamate dehydrogenase levels in the periportal and pericentral zone. The size of the periportal glutamate dehydrogenase-containing zone is appreciably smaller than that of carbamoylphosphate synthetase, whereas the pericentral glutamate dehydrogenase-containing zone is appreciably larger than that of glutamine synthetase. The heterogeneous distribution of glutamate dehydrogenase suggests the possibility that, under normal conditions, deamination of glutamate prevails in the periportal compartment and amination of glutamate in the pericentral compartment.

Mentré, P; Escaig, F

doi: 10.1177/36.1.3121721pmid: 3121721

A modification of the potassium pyroantimonate (PA) method for localization of calcium and sodium was tested using skeletal muscle of mouse. Massive diffusion of these cations, depending on the method of fixation, was demonstrated by analytical ion microscopy (AIM) images on the optical microscopy level. Rapid penetration of the fixative appeared to be the principal condition that reduced diffusion of Ca2+ and Na+. Paraformaldehyde (2%) appeared more efficient than glutaraldehyde (1%) for preserving metal composition. Addition of 1% phenol strikingly improved the quality of the AIM images. Supersaturated PA (4%) appeared to retain about 10 times more sodium in the tissue than insaturated PA (2%). The role of different buffers is also discussed, particularly collidine, which permitted better preservation of sodium. Fixation with this buffer should be very useful for study by AIM of large-scale distribution of sodium. These results are analyzed at the ultrastructural level in the accompanying report.

Mentré, P; Halpern, S

doi: 10.1177/36.1.3335770pmid: 3335770

A new formulation of the pyroantimonate (PA) method for localization of calcium and sodium is proposed and evaluated in mouse skeletal muscle. This study, performed at the ultrastructural level by means of transmission electron microscopy (TEM) and electron probe microanalysis (EPMA), completes a previous work done at the optical level with analytical ion microscopy (AIM), which enabled us to define the appropriate composition of fixatives. In our present experiments, calcium and sodium were shown localized in various cell structures, e.g., T-tubules, glycogen, granules, nuclei. For AIM, the best fixatives were characterized by PA supersaturation, which resulted in smaller crystals and a high rate of penetration in the presence of paraformaldehyde and either phenol or collidine. Contrary to the findings at the optical level, collidine did not give satisfactory results at the ultrastructural level. The method of floating sections on the microtome trough was an important cause of cation displacement. We found that alkalinization of the floating medium significantly decreased ion loss. The technique also provided an indication of the form of these elements: free or easily liberated cations were precipitated into coarse PA deposits; electron-positive chelates were "stained" by PA; neutral chelates were not stained, but some of them could be detected by EPMA. This PA method should make possible more precise localization of cellular calcium, such as in glycogen metabolism, and perhaps detection of movements of cytoplasmic calcium and sodium.

Cocco, L; Papa, S; Maraldi, N M; Santi, P; Martelli, A M; Rizzoli, R; Manzoli, F A

doi: 10.1177/36.1.3335771pmid: 3335771

We describe several morphological and functional modifications in isolated rat liver nuclei incubated in the presence of phosphatidylserine (PS) multilamellar vesicles (MLV). These effects, which occur through the release of histone H1, induce chromatin decondensation, as shown by electron microscopy and nuclease digestion. Flow cytometry was employed to monitor these changes in chromatin structure in isolated nuclei by means of perpendicular light scatter (PLS) and fluorescence signals. Chromatin decondensation induced by PS or by low pH treatment was accompanied by an increase in perpendicular light scatter and by less efficient binding of ethidium bromide. These flow cytometric findings are peculiar to chromatin decondensation induced by displacement of histone H1. Conversely, chromatin decondensation caused by lowering of the divalent ion concentration, without displacement of histone H1, is characterized only by an increase in perpendicular light scatter.

Singh, G; Singh, J; Katyal, S L; Brown, W E; Kramps, J A; Paradis, I L; Dauber, J H; Macpherson, T A; Squeglia, N

doi: 10.1177/36.1.3275712pmid: 3275712

Human lung lavage proteins were fractionated by centrifugation and molecular sieving. An antiserum to the post-albumin fraction of the soluble proteins reacted with a 10 KD protein and this protein was isolated by conventional chromatography. The protein, which has a pI of 4.8, consists of two 5 KD polypeptides and is rich in glutamic acid, leucine, serine, and aspartic acid amino acids. The protein does not bind to concanavalin A, pancreatic elastase, leukocyte elastase, or trypsin, and lacks anti-protease activity. It constitutes about 0.15% of the soluble proteins in lung lavage. Antibodies to the 10 KD protein specifically and exclusively stain Clara cells in human, dog, and rat. Staining of granules of Clara cells was prominent in the distal bronchioles; however, the non-ciliated cells of respiratory bronchioles did not stain for the 10 KD protein. This 10 KD protein appears in fetal lungs at 21 weeks of gestation, and was present in about 10% of the primary pulmonary adenocarcinomas. As a specific marker for Clara cells, this protein could be useful in the study of development, regulation of secretion, and pathobiology of these cells.