Journal of Histochemistry & Cytochemistry

Hogue-Angeletti, R; Xu, R Y; Gonatas, J O; Stieber, A; Gonatas, N K

doi: 10.1177/37.8.2666508pmid: 2666508

A monoclonal antibody, 3C9, has enabled the detection of a novel Golgi-specific protein in bovine tissues. Immunohistochemical studies at the light microscopic level have detected the 3C9 antigen only in certain cells: exocrine pancreas, gut epithelium, and thymus epithelium. Examination of gut and pancreas by immunoelectron microscopy showed a localization exclusive to the Golgi apparatus. The relative molecular weight of the antigen detected by immunoblotting is 210,000 daltons. The antigen is not extracted from microsomal membranes of bovine gut epithelium by sodium carbonate solutions. Furthermore, the 3C9 antigen enters into the detergent phase when Triton X-114 partitioning methods are used. These data strongly suggest that this novel antigen is an intrinsic membrane protein, resident in the Golgi apparatus of certain cells. Moreover, they enhance the hypothesis that the distribution of enzymes and polypeptides in the Golgi apparatus is cell specific.

Newman, G R; Jasani, B; Williams, E D

doi: 10.1177/37.8.2754250pmid: 2754250

While immunostaining serial semi-thin sections of acrylic resin-embedded normal human pituitary using antisera to human pituitary hormones, it became clear that several cells were stained by more than one antiserum. The tissue had been surgically excised from a patient with a prolactinoma. The tumor, which was immunoreactive only with antiprolactin antiserum, was distinctly different from the pieces of tissue under study which had normal pituitary architecture and demonstrated immunoreactivity with antisera against all six of the common pituitary hormones. A major immunoelectron microscopic investigation, using immunocolloidal gold and immunoperoxidase methods, revealed cells in which follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) were co-localized to the same electron-dense granules. Some similar cells also possessed electron-lucent granules immunoreactive only for anti-PRL antiserum. Adrenocorticotrophic hormone (ACTH) and PRL were also found in the same cell but were very largely localized to separate, morphologically different populations of electron-dense and -lucent storage granules. By employing double immunolabeling, a few granules in the ACTH/PRL cells were shown to be immunoreactive to both anti-ACTH and anti-PRL antisera. The possibility that the multipotential stem cells is discussed.

Wide, M; Persson, H; Gunnarsson, S; Wide, L; Seifi, L

doi: 10.1177/37.8.2474022pmid: 2474022

We describe two different techniques with plastic embedding in in situ hybridization histochemistry (ISHH). Their applicability was demonstrated by use of human placenta of the tenth gestational week and a tritium-labeled cDNA probe for the beta-subunit of hCG. In the first method, ISHH was performed on whole pieces of tissue (en bloc ISHH) pretreated with a weak acid solution, embedded in methacrylate, and sectioned at 3 microns for autoradiography. In the second technique, en bloc ISHH was carried out on tissue pre-treated with the weak acid and thereafter with detergent to further facilitate probe penetration. An acrylic resin was used for embedding, and section thickness was reduced to 1 microns. With both techniques, beta hCG cDNA/mRNA hybrids were localized exclusively to the syncytiotrophoblast (ST), in agreement with a previous study using sections of frozen placentas for hybridization to the same probe. However, owing to the higher resolution of the plastic sections the reliability of this localization was greatly increased. The number of autoradiographic grains over the acrylic resin 1-microns sections was found to be considerably higher than that over the methacrylate 3-microns sections. This study showed that treatment of tissue with detergent before en bloc ISHH, with subsequent embedding in acrylic resin and sectioning at 1 microns, gives high resolution in combination with a high signal-to-noise ratio after autoradiography. As the acrylic resin permits cutting of ultrathin sections, the results suggest that the technique may become useful for ISHH studies at the subcellular level.

Fauquet, M; Ziller, C

doi: 10.1177/37.8.2569003pmid: 2569003

Catecholamine (CA) synthesis is one of the phenotypic traits expressed by some neural crest-derived cells in vivo and in vitro. In the present study, we have evidenced, in quail embryos, the expression of the first enzyme of CA metabolism, tyrosine hydroxylase (TOH), using a monoclonal antibody raised against the quail enzyme. This antibody also recognizes TOH from chick and pleurodele, but not from several mammalian species (rat, human). We have also investigated the extent to which TOH-positive cells, differentiated in neural crest cultures, express structural neuronal markers and display vasoactive intestinal polypeptide (VIP) and substance P (SP) immunoreactivity. Double-immunolabeling experiments show that, in vitro, half of the population of TOH-positive cells exhibits tetanus toxin binding sites but none of them are recognized by a neurofilament antibody. On the other hand, some TOH-positive cells contain VIP or SP. These observations suggest that under our culture conditions autonomic neural crest precursors differentiate only into immature sympathoblasts, but are able to synthesize peptides in addition to CA.

Pekki, A; Tuohimaa, P

doi: 10.1177/37.8.2754251pmid: 2754251

We describe a new application of freeze-drying and vapor fixation for immunohistochemical location of soluble proteins. The method avoids the liquid phase, which eliminates the possible diffusion of soluble proteins. Two vapor fixatives, paraformaldehyde and p-benzoquinone, were tested and p-benzoquinone was found to preserve antigenicity of progesterone receptor (PR) and ovalbumin better than paraformaldehyde. The method proved to be highly sensitive, since higher concentrations of antigen were found in some tissues and some tissues found to be antigen negative by earlier liquid fixation methods proved to contain antigen. The location of PR as a highly soluble protein was studied. With the present method, both unoccupied and occupied PR were located in the nuclei, a similar finding as with the earlier liquid fixation method. The results further support the concept that PR is an intranuclear protein independent of its ligand occupation. PR was detected in a few cells inside the follicles of the bursa of Fabricius and in the smooth muscle cell nuclei of the small intestine, observations not previously made owing to the insensitivity of the earlier methods.

Hazlett, L D; Mathieu, P

doi: 10.1177/37.8.2754252pmid: 2754252

The purpose of this study was to develop a procedure to quantitatively examine corneal epithelial apical cell membrane-associated glycoconjugates. Saccharide moieties on young, mature, and aged corneal epithelial cells were detected and localized in corneas of immature and adult mice by using colloidal gold-labeled lectins and transmission electron microscopy (TEM). In general, dense binding to the corneal epithelial apical surface cell membranes with wheat germ agglutinin (WGA) was seen in the adult, whereas the immature cornea bound less WGA-gold. Neuraminidase digestion decreased binding of the conjugate on epithelial plasma membranes of young and mature cells in adult cornea. Lectin-gold binding was decreased in the immature cornea on mature and aged cells. WGA-gold binding after neuraminidase was elevated on young cells of immature and on aged cells of adult animals. No binding of peanut agglutinin (PNA) or horse gram agglutinin (DBA) to the corneal epithelial surface was seen in animals of either age. After neuraminidase digestion, PNA binding sites were exposed only on the adult corneal surface. These data suggest that a terminal trisaccharide sequence, sialic acid-galactose beta(1----3)-N-acetylgalactosamine, is present at the adult corneal surface but is absent or at undetectable levels at the corneal surface of the immature animal. These data may be of significance in light of the dissimilar pattern of P. aeruginosa recognition and binding to the immature vs adult corneal epithelium.

Hartman, A L; Sawtell, N M; Lessard, J L

doi: 10.1177/37.8.2754253pmid: 2754253

A minimum of six very similar but distinct actin isoforms are encoded by the mammalian genome. Developmental regulation of these genes results in a tissue-specific distribution of the isoforms in the adult. Using a panel of actin specific monoclonal antibodies (MAb), we recently reported the expression of two unique actin isoforms in adult rat intestinal brush border. In this report, we examine the developmental expression of these and other actin isoforms in rat intestinal epithelial cells. Isoforms containing the HUC 1-1 and/or C4 epitopes are present by day 15 of gestation and are continuously expressed throughout adult life. Unexpectedly, the gamma-enteric smooth muscle isoactin, defined by the B4 epitope, is transiently expressed in these non-muscle cells late in gestation. The alpha-vascular smooth muscle isoform, however, is not expressed in intestinal epithelial cells during development and, as previously reported, both smooth muscle isoforms are absent in epithelial cells of adult intestine. In addition, we demonstrate that although multiple isoforms are expressed simultaneously in these cells, they are not uniformly distributed at the subcellular level, suggesting that the cell recognizes the actin isoforms as functionally distinct entities.

Watson, L P; Kang, Y H; Falk, M C

doi: 10.1177/37.8.2526836pmid: 2526836

The interactions of osteoblasts with one another and with the extracellular milieu are of vital importance for cell function. These interactions are mediated by cell membrane-associated components. In the present work, we studied the distribution of several mediators known to be associated with the cell surface, using ultrastructural cytochemistry, to characterize the three cell membrane domains (osteoid, lateral, and vascular) of osteoblasts. Osteoblasts in neonatal rat calvariae were studied for cell surface distribution of alkaline phosphatase (APase), calcium-activated adenosine triphosphatase (Ca2+-ATPase), calcium, soybean agglutinin (SBA)-reactive sites, and peanut agglutinin (PNA)-reactive sites. APase was absent in the osteoid domain but was evenly distributed in the other domains. Ca2+-ATPase was found to be concentrated mainly in the lateral domains. In contrast, calcium was present in all cell membrane domains. Using lectins conjugated to horseradish peroxidase, we demonstrated that SBA binding sites were evenly distributed along the osteoblast cell membrane, whereas PNA binding sites were absent or minimally present in the osteoid and lateral domains but were evenly distributed in the vascular domain. These results suggest that the various functions of osteoblasts may be facilitated by specialized cell membrane domains which are cytochemically distinct. Previous reports have failed to demonstrate the cytochemical differences between the three domains of the osteoblast cell membrane.

Aiso, S; Yasuda, K; Shiozawa, M; Yamamoto, H; Sogo, T

doi: 10.1177/37.8.2754254pmid: 2754254

Glutathione S-transferase (GST) EC 2.5.2.18) catalyzes conjugation of reduced glutathione with hydrophobic substrates, such as S-epoxide active molecules. It participates in glutathione metabolism and the gamma-glutamyl cycle, playing an important role in detoxification and biosynthesis of many compounds. It is also known as a marker of pre-neoplasia in chemical hepatocarcinogenesis. Isoelectric focusing studies have revealed that this enzyme is composed of several isozymes, one of which, an acidic form of GST called GST-pi, has been extracted from human placenta. In this study, we prepared monoclonal antibodies (MAb) against human GST-pi from placenta. Specificity was confirmed by immunoblots of GST-pi after polyacrylamide gel electrophoresis and inhibition testing of enzyme activity by the antibody. The subclass of the antibody was IgG1 and the light chain was kappa. In light microscopic immunohistochemical studies of human placenta using the MAb, GST-pi was localized diffusely in the cytoplasm and along the apical cell membranes of syncytial cells in villi and in the cytoplasm of cytotrophoblastic cells in the basal plate. The MAb we prepared may also be useful for analyzing the enzyme's function in detoxification and biosynthesis of many compounds, as well as for oncological studies, such as diagnosis of malignant disease and localization of oncofetal proteins in malignant tissues.

Sokol, R Z; Wang, X S; Lechago, J; Johnston, P D; Swerdloff, R S

doi: 10.1177/37.8.2666509pmid: 2666509

We identified relaxin in human male prostate by use of an anti-human relaxin analogue polyclonal antibody and the avidin-biotin-immunoperoxidase method. The antibody was obtained by immunizing a rabbit with a synthetic human relaxin analogue which has 95% sequence homology with native human relaxin. Human prostate tissues incubated with the anti-human relaxin analogue exhibited positive immunostaining up to an antibody dilution of 1:3200. Inhibition of immunostaining with this antibody by excess relaxin analogue demonstrated specificity of the antibody. The exact role of relaxin in human male reproductive physiology remains to be fully elucidated.