Journal of Histochemistry & Cytochemistry

Testillano, P S; Gorab, E; Risueño, M C

doi: 10.1177/42.1.7505298pmid: 7505298

We describe a new ultrastructural method for locating transcription on ultra-thin sections. The use of anti-DNA/RNA hybrid antibodies provides specific labeling on precise structures of the nuclear compartments of several cell types. All mammalian and plant material studied (HeLa cells, lymphocytes, onion root meristematic cells) showed the same pattern of labeling: fibrillar structures in the interchromatin region and discrete regions of the dense fibrillar component at the periphery of the fibrillar centers in the nucleolus. The specificity of the immunogold labeling was tested by RNAse H digestion and by pre-blocking the antibody with synthetic DNA/RNA hybrids; in both cases no gold particles were observed. This method has considerable advantages compared with current techniques, constituting a very useful tool to map transcriptionally active loci in a variety of cells.

Vila-Porcile, E; Picart, R; Tougard, C

doi: 10.1177/42.1.8263322pmid: 8263322

We used the reverse hemolytic plaque assay (RHPA), a method for detecting secretion by single cells, to demonstrate functional heterogeneity among prolactin (PRL) cells of rat anterior pituitary at the light microscopic (LM) level. We attempted to adapt RHPA for electron microscopy (EM) to define the relationships between fine structure and secretory activity in individual PRL cells. A major modification of the technique, intended to improve preservation of ultrastructure, was allowing the cells to recover after their enzymatic dispersion from the pituitary tissue, through a brief (3-4 hr) culture step before the assay. Adaptation for EM was achieved by the use of plastic slides for construction of specialized Cunningham chambers, permitting application of all the EM procedures (flat embedding, punching of selected areas) usually employed for cultured pituitary monolayers. Moreover, immunocytochemical pre- and post-embedding methods were also applied for cell identification and study of subcellular hormone distribution. Such a modified RHPA enabled us to analyze the ultrastructure of plaque-forming cells surrounded by their companion red blood cell ghosts. The first results with EM RHPA showed that under basal conditions a subpopulation of PRL cells containing small granules (150-200 nm) was actively secreting, whereas PRL cells with large (300-600 nm) and irregular granules appeared to be stimulated by thyrotropin-releasing hormone or KCl. The EM RHPA technique described here might receive more general application and could be utilized for study of many other secretory systems.

Markovic, N; Markovic, O; Roberts, J; Markovic, S

doi: 10.1177/42.1.7903327pmid: 7903327

We developed a new cytochemical assay for identification of cells containing inosine monophosphate dehydrogenase (IMPDH) and measurement of tumor cell sensitivity to the escalating dose/schedule of the IMPDH pattern-targeting drugs. The assay is based on cytochemical principles for development of DH activity markers inside morphologically classified cells, image analysis for measuring the amount of this marker, and computer assistance for data management. The assay was optimized on a human leukemia cell line (K562-NS) and reference values for enzyme activity were established. Assay specificity was determined with different substrates and enzyme inhibitors. Sensitivity depends on the measuring instrument (image analyzing system), and the precision of the biological model used for assessment of reference values was high. IMPDH-positive malignant cells were found in all specimens obtained from acute leukemia and solid tumor patients (13/13 and 29/29) and in four human tumor cell lines (K562, K562-NS, HL60, and HL60-M). Cells of the K562-NS line were exposed to tiazofurin and ribavirin in conventional assays for assessment of drug-induced acute and subacute toxicity/sensitivity. A reduction of IMPDH activity was recorded at drug concentrations below the range at which cell damage appeared.

Larsson, L I; Houggaard, D M

doi: 10.1177/42.1.7903328pmid: 7903328

Gastrin is produced by gastric endocrine cells and regulates acid secretion and mucosal cell proliferation. Somatostatin inhibits gastrin secretion, and exogenous somatostatin was recently reported to decrease gastrin gene transcription and to reduce gastrin mRNA stability. Somatostatin cells extend cytoplasmic processes that contact gastrin cells and other cells. Using quantitative in situ hybridization combined with immunocytochemistry, we demonstrate that gastrin cells in close contact (< or = 0.2 micron) with somatostatin cells express significantly lower levels of gastrin mRNA than other gastrin cells. Our data show that cells with high levels of gastrin mRNA and no detectable contacts predominate at the base of the glands. Hence, the results provide evidence that endogenous somatostatin regulates gastrin gene expression in a subpopulation of gastrin cells and point to regulatory heterogeneity of these cells. This represents the first direct microscopic evidence for paracrine gene regulation. The new quantitative double-staining approach may be generally useful for studies of cell interactions and cell sociology.

Watson, E L; Oliver, C; D'Silva, N; Belton, C M

doi: 10.1177/42.1.7505300pmid: 7505300

The GTP-binding protein G(o) was localized immunohistochemically in the rat parotid gland and in other exocrine glands with specific G(o) antibodies. Immunohistochemical studies revealed that affinity-purified G(o alpha) polyclonal antibody (GO/85) immunoreacted primarily with duct cells of the rat parotid gland; immunoreactivity was also noted in duct cells of the rat submandibular, mouse parotid, and mouse submandibular glands. Light labeling of rat parotid and submandibular gland acinar cells was also noted. G(o alpha) antiserum (9072) differing in specificity for epitopes within G(o alpha) produced similar results. This antiserum also immunoreacted with rat submandibular duct cell secretory granule membranes. In contrast, in rat and mouse pancreas G(o alpha) antibodies immunoreacted primarily with islet cells. Duct cells were negative but there was light labeling of rat pancreatic acinar cells. The apparent duct specificity of G(o alpha) staining was further verified by demonstrating that G(o alpha) antibodies immunoreacted with HSG-PA cells, a human transformed salivary duct cell line. Specificity in immunohistochemical labeling of HSG-PA cells was confirmed by Western blot analysis. The results demonstrate that G(o) appears to be selectively expressed in the duct cells of rat parotid gland and other salivary glands. The selective enrichment of G(o) in duct cells suggests that this G-protein plays an important role in duct cell physiology.

Anderson, K D; Karle, E J; Reiner, A

doi: 10.1177/42.1.7505301pmid: 7505301

For many neural regions it is of interest to know the identity of the target structures of two different types of inputs to that neural region. Such studies require use of a triple-label immunohistochemical method to differentially label the class of target structure and the two types of input so that they can be visualized at the electron microscopic (EM) level. We describe here a procedure for combining three different markers (diaminobenzidine, benzidine dihydrochloride, and silver-intensified immunogold) for triple-label EM immunohistochemical pre-embedding labeling. All three markers are distinct at the LM and EM levels. An example of this approach as applied to studying striatal input to the ventral tegmental area is presented and the advantages of this approach are discussed.

Schumacher, U; Adam, E; Hauser, F; Probst, J C; Hoffmann, W

doi: 10.1177/42.1.7903329pmid: 7903329

The purpose of this study was to investigate the structure and chemical composition of the mucous skin gland of Xenopus laevis by combined morphological and biochemical techniques. Protein backbones of mucins were localized immunohistochemically in the gland with anti-peptide antibodies. Acid mucins were demonstrated by conventional histochemical techniques and their terminal carbohydrate residues were localized by lectin histochemistry. A close correlation between antibody and lectin binding of the same glycoproteins was achieved on Western blots from isolated skin gland mucins, indicating that the lectin-binding sites were due to defined mucin molecules. The cone cell, thought to be a degenerative cell in the past, contained mucin granules with an electron-dense core, strong PAS reactivity, a special lectin-binding pattern, and localization of integumentary mucins FIM-B.1 and FIM-C.1. These results indicate that cone cells are a distinct cell type, elaborating and releasing particular mucins, and that functional heterogeneity of mucus-producing cells exists in the mucous skin glands of X. laevis.

Velkeniers, B; Vergani, P; Trouillas, J; D'Haens, J; Hooghe, R J; Hooghe-Peters, E L

doi: 10.1177/42.1.8263325pmid: 8263325

Cells expressing IL-6 mRNA were detected by in situ hybridization in normal pituitaries. In normal untreated rat pituitary the expression was very low. Within hours after IP administration of liposaccharide, IL-6 mRNA accumulated in the anterior lobe of the pituitary. Production of IL-6 was monitored after dissociation and culture of pituitary cells. High levels (8000 pg/ml) were recovered after 72 hr in culture. In normal human pituitaries, less than 1% of cells expressed IL-6 mRNA or IL-6 receptor mRNA (IL-6-R mRNA). In gonadotropinomas, prolactinomas, and non-functioning adenomas, only rare, scattered positive cells were found for either IL-6 or IL-6-R. In contrast, both genes were highly expressed in ACTH- and GH-secreting tumors at the junction of adenoma and infiltrating fibrous tissue and around blood vessels. The combined expression of IL-6 and IL-6-R suggests that IL-6 acts in an autocrine or in a paracrine way in ACTH and GH adenomas.

Pedrosa-Domellöf, F; Thornell, L E

doi: 10.1177/42.1.8263326pmid: 8263326

We studied serial sections of human fetal limb muscles (10-25 weeks of gestation) by light microscopic (LM) immunocytochemistry, using specific antibodies against slow-tonic, slow-twitch, fetal, embryonic, and alpha-cardiac myosin heavy chain (MHC) isoforms, neurofilament protein, laminin, and myomesin. One set of the first-generation myotubes expressed slow-tonic MHCs, and slow-twitch, fetal, and embryonic MHCs from the tenth week of gestation. These primary myotubes were identified as developing nuclear bag fibers. Second-generation myotubes in close apposition to the primary nuclear bag myotubes initially expressed only fetal and embryonic MHCs. One or more of these secondary myotubes acquired expression of slow-tonic and slow-twitch MHCs and gave rise to nuclear bag fibers. Most of the nuclear bag precursors expressed alpha-cardiac MHC. The secondary myotubes that expressed fetal and embryonic MHC but not slow-tonic, slow-twitch, or alpha-cardiac MHCs gave rise to the nuclear chain fibers. This study shows that different populations of fiber precursors, each with a unique sequence of MHC expression, gave rise to the nuclear bag and chain fibers, despite the presence of a common afferent nerve, from the early stages.

van den Born, J; van den Heuvel, L P; Bakker, M A; Veerkamp, J H; Assmann, K J; Berden, J H

doi: 10.1177/42.1.8263327pmid: 8263327

We raised monoclonal antibodies (MAb) against the core protein and the heparan sulfate (HS) side chain of heparan sulfate proteoglycan (HSPG) from glomerular basement membranes (GBM). Anti-HSPG-core MAb were obtained after immunization of mice with HSPG purified from human GBM and the anti-HS MAb after immunization of mice with HSPG from rat glomeruli, which crossreacted with human HS and GBM HSPG. The specificity of the MAb was demonstrated by ELISA studies, Western blotting, inhibition experiments, and indirect immunofluorescence (IF) on kidney cryostat sections pre-treated with glycosaminoglycan (GAG)-degrading enzymes. Indirect IF on normal human kidney tissue showed prominent GBM staining for both MAb, with variable staining of the other renal basement membranes (BMs). By indirect immunoelectron microscopy (IEM), most intense staining was observed at the endothelial side of the GBM for both MAb, although the staining patterns were not identical. Both MAb were used to localize HSPG in human tissues by indirect IF. They bound to antigens present in the BMs of most tissues examined, including those of epithelia and endothelia. Differences between both MAb were observed for BMs of muscle cells, since the anti-HSPG core protein MAb (JM-72) staining was negative, whereas the anti-HS MAb (JM-403) clearly stained these structures. Comparison of our staining patterns in human tissues with the distribution of other anti-BM HSPG antibodies suggests that there are at least two types of BM HSPG, which have common epitopes on the HS side chains recognized by JM-403.