Journal of Histochemistry & Cytochemistry

Ozden, S; Aubert, C; Gonzalez-Dunia, D; Brahic, M

doi: 10.1177/38.7.1693934pmid: 1693934

We used 35S-labeled and biotinylated cRNAs (riboprobes) to detect simultaneously two different mRNAs by in situ hybridization. In a first step we established the conditions under which each type of probe achieved the same high level of sensitivity. We then used these conditions to hybridize BHK cells infected with Theiler's virus, a murine picornavirus, with a mixture of a virus-specific biotinylated riboprobe and a 35S-labeled riboprobe specific for beta-actin mRNA. Both mRNAs could be detected in the same cell, although the sensitivity achieved by the radiolabeled probe was reduced by about 40% by the simultaneous hybridization with the biotinylated probe.

Tuson, J R; Pascoe, E W; Jacob, D A

doi: 10.1177/38.7.2355173pmid: 2355173

We describe a novel immunohistochemical technique which permits the detection of specific binding of human monoclonal antibodies (MAb) to cryostat sections of human tissues. The technique overcomes the problem of background staining caused by the presence of endogenous immunoglobulins in tissue sections. This is achieved by the formation of a molecular complex of the primary antibody (a human MAb), horseradish peroxidase-conjugated goat anti-human immunoglobulin, and normal human serum. This complex is then incubated with cryostat sections of human tissue, and binding of the complex is demonstrated using diaminobenzidine/hydrogen peroxide. The method is suitable for immunohistochemical screening of small samples of tissue culture supernatant for the presence of human MAb of potential interest, and for determining the pattern of binding of such MAb to a wide range of normal and pathological human tissues.

Nishimura, H; Nishimura, N; Tohyama, C

doi: 10.1177/38.7.2355174pmid: 2355174

We studied the immunohistological localization of metallothionein (MT), a low molecular weight metal binding protein, in male rat genital organs (testis, epididymis, ejaculatory duct, seminal vesicle, coagulating gland, and prostate) by use of the avidin-biotin-peroxidase complex method. MT concentrations in testis, seminal vesicle, and prostate ranged from 15-30 micrograms/g tissue. In testis, seminiferous tubules with mature spermatozoa exhibited weak MT staining, whereas the tubules containing differentiating spermatogenic cells but not containing spermatozoa showed strong MT staining. No MT immunostaining was observed in Leydig cells. In growing rat testes, the pattern of MT immunostaining was found to change with development: MT was found in supporting cells only on Day 7, spermatogonia adjacent to basement membrane on Day 14, and spermatocytes localized in the central part of the tubules on Day 21. Strong MT immunostaining in the basal cells was a common feature in other genital tissues, except the ductus efferentes. In prostate, the strongest MT staining was found in the lateral lobe, and MT was localized in apocrine secretions in the dorsal lobe. The present results suggest a close association of MT with cell proliferation and differentiation, as well as possible involvement of MT in supply or storage of zinc ions.

Renegar, R H; Southard, J N; Talamantes, F

doi: 10.1177/38.7.2355175pmid: 2355175

Two hormones with lactogenic activity are produced by the hamster placenta during the second half of pregnancy. One of these hormones, hamster placental lactogen II (haPL-II), has been well characterized; however, its cellular source is not known. In the present study, haPL-II was localized in placental tissues using a specific antibody and the avidin-biotin-peroxidase immunohistochemical technique. Because relaxin has been localized in the hamster placenta, it was of interest to determine if haPL-II and relaxin are localized in the same cells. haPL-II immunoactivity was observed in primary and secondary giant trophoblast cells of the placenta on Days 12, 14, and 15 of pregnancy. On Day 15 positive staining was also observed in large cells located within mesometrial arteries and in eosinophilic bodies associated with degenerating sheathed arteries of the decidua basalis. haPL-II-positive staining was not observed in placentae from Days 8 or 10 of pregnancy. On Day 14, haPL-II was colocalized with relaxin in 75% of the giant trophoblast cells observed. Therefore, it is probable that these hormones are synthesized and secreted by the same cell.

Parkkila, S; Kaunisto, K; Rajaniemi, L; Kumpulainen, T; Jokinen, K; Rajaniemi, H

doi: 10.1177/38.7.2113069pmid: 2113069

Human salivary carbonic anhydrase (HCA VI) was purified by inhibitor affinity chromatography and its location in the human parotid and submandibular glands identified, using a polyclonal antiserum raised against the purified enzyme in rabbits in conjunction with the peroxidase-antiperoxidase complex method. The antibodies raised against the purified enzyme in rabbits did not crossreact with the HCA II or I. However, they slightly recognized human IgA; the antiserum was therefore absorbed with human IgA before immunohistochemical use. HCA VI-specific staining was detected in the cytoplasm and particularly in the secretory granules of the serous acinar cells of both parotid and submandibular glands, the staining of the secretory granules being most distinct in paraformaldehyde-fixed tissues. Some epithelial cells and the luminal content of the striated ducts also gave a specific HCA VI staining. Staining specific for HCA II was also found in the granules of the serous acinar cells, particularly in the submandibular gland when Carnoy fluid fixation was used. Slight HCA II-specific staining was also detected in the striated ductal cells in the Carnoy fluid-fixed specimens. No staining specific for HCA I was detected. The results indicate that the serous acinar cells in human parotid and submandibular glands contain abundant HCA II and HCA VI. Interestingly, only HCA VI is secreted into the saliva, although both enzymes appear to be located in structures resembling the secretory granules in the acinar cells. The enzymes probably form a mutually complementary system regulating the salivary buffer capacity.

Scammell, J G; Rosa, P; Hille, A; Huttner, W B

doi: 10.1177/38.7.2192000pmid: 2192000

GH4C1 cells are a rat pituitary tumor cell strain in which the level of cellular prolactin (PRL) and PRL-containing secretory granules can be regulated by hormone treatment. The chromogranins/secretogranins (Sg) are a family of secretory proteins which are widely distributed in the secretory granules of endocrine and neuronal cells. In the present study, we investigated in GH4C1 cell cultures the regulation of the cell content of the Sg by immunoblotting and the relationship between the storage of Sg I and Sg II and PRL by double immunocytochemistry. GH4C1 cells grown in the presence of gelded horse serum, a condition in which these cells contain a low level of secretory granules, contained low levels of PRL, Sg I, and Sg II. Treatment of GH4C1 cells with a combination of 17 beta-estradiol, insulin, and epidermal growth factor for 3 days, known to induce a marked increase in the number of secretory granules, increased the cell contents of PRL, Sg I, and Sg II. To determine whether the induction of PRL was morphologically associated with that of the Sg, the distribution of PRL and the Sg was determined by double immunofluorescence microscopy. After hormone treatment, 54% of cells showed positive PRL immunoreactivity, fluorescence being extranuclear and consistent with staining of the Golgi zone and secretory granules. Forty-six percent of PRL-positive cells stained coincidently for Sg I, while 72% of the PRL cells were also reactive with anti-Sg II. To determine whether PRL storage was associated with storage of at least one of the Sg, cells were stained with anti-PRL and anti-Sg I and anti-Sg II together. Eighty-six percent of PRL cells stained for one or the other of the Sg. Therefore, PRL storage in GH4C1 cell cultures is closely but not completely associated with the storage of Sg I and/or II.

Croul, S; Mezitis, S G; Stieber, A; Chen, Y J; Gonatas, J O; Goud, B; Gonatas, N K

doi: 10.1177/38.7.2355176pmid: 2355176

We used a monoclonal antibody (10A8), derived from mice immunized with fractions enriched in Golgi apparatus of rat brain neurons, to isolate an intrinsic membrane sialoglycoprotein of 160 KD from rat brain. By immunoelectron microscopy the sialoglycoprotein, named MG-160, was localized in medical cisternae of the Golgi apparatus of neurons, glia, adenohypophysis, and cultured rat pheochromocytoma (PC 12). The monoclonal antibody (MAb) reacted only with rat tissues. Because the epitope(s) recognized by a monoclonal antibody may be restricted, localization of an antigen by a single MAb may not reflect the extent of the distribution of antigen in various species and tissues. Therefore, to further investigate the presence and localization of MG-160 or of an antigenically related protein in several species and tissues, we used a polyclonal antiserum raised against MG-160 purified by antibody (10A8) affinity chromatography. Immunoblots of crude microsomal fractions from rat brain probed with the antiserum against MG-160 showed two to three prominent bands of approximately 160, 150, and 68 KD. Immunoblots of crude microsomal fractions from human, chicken, and frog brains showed prominent bands of 130-140 and 68 KD. Immunoblots of crude membrane fractions from Saccharomyces cerevisiae showed prominent bands of approximately 110-120 and 80 KD. Light microscopic immunocytochemical studies with frog, chicken, mouse, rat, rabbit, bovine, and human brains and with several other rat and human tissues showed a staining pattern consistent with the Golgi apparatus. Immunoelectron microscopy with rat and human brain and with rat myocardium and pituitary showed prominent and exclusive staining of cis, medial, and occasionally trans cisternae of the Golgi apparatus. The cisternae of the trans Golgi network were not stained. These findings are consistent with the hypothesis that a polypeptide related to MG-160 is present in the Golgi apparatus of several tissues in human, rodents, chicken, and frog and possibly in Saccharomyces cerevisiae. The antiserum to MG-160 represents a reliable reagent for immunohistochemical visualization of the Golgi apparatus in brain and several other human tissues obtained at autopsy, fixed with Bouin's, and embedded in paraffin.

Michel, E; Parsons, J A

doi: 10.1177/38.7.2355177pmid: 2355177

We studied prolactin (PRL) binding sites on Nb2 lymphoma cells using two different light microscopic methods. First, histochemical detection was accomplished by using an aminomethyl coumarin-acetic acid-conjugated ovine prolactin molecule (AMCA-oPRL) on both glutaraldehyde-fixed and unfixed Nb2 lymphoma cells. Binding of AMCA-oPRL was studied after UV illumination and appeared as punctate fluorescence associated with many but not all cells. Binding was abolished when tissue sections were treated with excess unlabeled lactogenic hormones and was unchanged when a non-lactogenic hormone was used for displacement. Counting revealed significant differences between the number of labeled cells in populations known to exhibit up- or down-regulated PRL receptors. Second, indirect immunocytochemistry of Nb2 PRL receptors was accomplished by immunological detection of exogenously added ovine PRL using two antisera directed against ovine PRL. Visualization of the ligand-antibody complexes was accomplished by confocal laser scanning microscopy. Staining was restricted to a subpopulation of cells. The morphological results presented here add to the previous physiological and biochemical data on the presence of lactogenic hormone receptors on Nb2 lymphoma cells.

Sahara, N; Siraganian, R P; Oliver, C

doi: 10.1177/38.7.1693935pmid: 1693935

RBL-2H3 cells have been widely used to study histamine release in vitro. It was previously shown that these cells undergo striking morphological changes after IgE-mediated secretion. The present study was undertaken to examine if the morphological changes were dependent on activation of the Fc epsilon receptor. Therefore, the cells were stimulated to release histamine by two different mechanisms: activation of the Fc epsilon receptor by antigen and treatment with the calcium ionophore A23187. Cell surface and cytoskeletal changes were examined by fluorescence microscopy and scanning electron microscopy after either IgE- or ionophore-mediated histamine release. After exposure of the cells to either secretagogue, the cells spread over the surface of the culture dish and underwent rearrangement of the cytoskeleton. In addition, scanning electron microscopy revealed that deep ruffles developed on the surface of the cells undergoing IgE-mediated release. The surface changes were not as pronounced with the ionophore. The distribution of the cytoskeletal elements was examined by immunofluorescence using FITC-phalloidin and antibodies against vimentin and tubulin. In unstimulated cells actin was localized at the cell periphery, just under the plasma membrane. In the stimulated cells it was associated with the cell periphery and concentrated in the surface ruffles. As the stimulated cells spread, intermediate filaments and microtubules became distributed throughout the cell body, but there was no obvious association with the membrane ruffles. These morphological changes were dependent on the presence of extracellular calcium and on the concentration of ionophore or antigen, and were also correlated with the amount of histamine released. Additionally, IgE-mediated stimulation led to increased uptake of the soluble-phase tracer Lucifer yellow, whereas stimulation with the ionophore A23187 showed no increase in Lucifer yellow internalization. Ionophore A23187 produced changes similar but not identical to those seen in the RBL-2H3 cells after IgE-mediated histamine release. The differences may be owing to the involvement of the Fc epsilon receptor in IgE-mediated secretion.

Yamada, K; Abe, Y; Fujimori, O

doi: 10.1177/38.7.2355178pmid: 2355178

We have established an efficient method for the histochemical demonstration of protein-bound amino groups by light microscopy, using a ninhydrin or alloxan-thiocarbohydrazide-silver proteinate (NHY or ALX-TCH-SP) sequence followed by a physical development (PD) procedure. As a result of the present experimental studies on Carnoy's solution-fixed paraffin sections of a series of rat tissues, including three types of major salivary glands, liver, pancreas, stomach, duodenum, jejunum, colon, kidney, prostate, and spleen, the sensitivity and specificity of the new method were found to be sufficient. In the tissues tested, protein-bound amino groups were visualized by distinct brownish or blackish reaction products. Comparisons of the particular method with the NHY or ALX-Schiff methods employed hitherto have substantiated the fact that the former method leads to apparently higher visibility of reaction products than the latter.