Showing papers in "Journal of Histochemistry and Cytochemistry in 1989"

Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation.:

Abstract: The binding of Congo red to several purified amyloid-like peptides having a beta-pleated sheet conformation was quantitatively examined. Congo red binds preferentially to the beta-pleated sheet conformation of both insulin fibrils and poly-L-lysine. Congo red does not bind nearly so well to poly-L-serine or polyglycine, despite the fact that these peptides also have a beta-pleated sheet conformation. Binding to insulin fibrils was saturable with an apparent Bmax of 2 moles of Congo red per mole of insulin fibrils and an apparent KD of 1.75 x 10(-7) M. Binding to beta-poly-L-lysine was similar but had a much higher apparent Bmax of 43. Binding of Congo red to beta-poly-L-lysine was pH dependent and appeared to be determined by the number of protonated lysine residues in the 250 amino acid peptide. We present a new hypothesis in which Congo red binds to amyloid-like proteins via bonds between the two negatively charged sulfonic acid groups of Congo red and two positively charged amino acid residues of two separate protein molecules which are properly oriented by virtue of the beta-pleated sheet conformation of the peptide backbone.

Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein.

Abstract: Using peroxidase immunohistochemistry, we examined the distribution of P170, a multidrug transport protein, in normal tissues by use of two different monoclonal antibodies (MAb). MAb MRK16 is a MAb that has been shown to react with an epitope in P170 located on the external face of the plasma membrane of multidrug-resistant human cells. MAb C219 has been shown to react with P170 in many mammalian species, and detects an epitope located on the cytoplasmic face of the plasma membrane. Using MRK16, we have previously described the localization of P170 on the bile canalicular face of hepatocytes, the apical surface of proximal tubular cells in kidney, and the surface epithelium in the lower GI tract in normal human tissues. In this work, we report that MRK16 also detects P170 in the capillaries of some human brain samples. A similar pattern was found using MAb C219 in rat tissues. in addition, MAb C219 showed intense localization in selected skeletal muscle fibers and all cardiac muscle fibers in rat and huma...

Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and anti-chymase antibodies.

Abstract: We developed an improved immunohistochemical technique for distinguishing human mast cells of the MCT (tryptase-positive, chymase-negative) and MCTC (tryptase-positive, chymase-positive) types utilizing a biotinylated murine anti-chymase monoclonal antibody (MAb), termed B7, and an alkaline phosphatase-conjugated murine anti-tryptase MAb, termed G3. The B7 MAb also was used to show the selective presence of chymase in mast cells. The distribution of MCT and MCTC cells in Carnoy's fluid-fixed tissue sections of human lung, skin, small intestine, and tonsils was analyzed by the new technique and the results compared to those obtained with the older method using a rabbit polyclonal antichymase antibody and a mouse anti-tryptase MAb in indirect immunoperoxidase and indirect immunoalkaline phosphatase protocols, respectively. In tissues known to contain predominantly mature mast cells, there were no quantitative differences between the two techniques, although the staining intensity achieved with the anti-chymase MAb was greater and without development of high background, compared to results achieved with the polyclonal antibody. MCT cells were the predominant type seen in the alveoli of the lung (93%) and in the small intestinal mucosa (81%). MCTC cells predominanted in the skin (99%) and in the small intestinal submucosa (77%) and, to a lesser degree, in tonsils (60%). However, in newborn foreskin tissue which contains predominantly immature forms of mast cells, 75% of all mast cells were stained uniformly and intensely with B7, whereas only 43% were stained with the polyclonal anti-chymase antibody. Therefore, the use of MAb provides for better standardization of reagents and more accurate assessment of the distribution of human MCT and MCTC cells in tissues than previously available methods.

Alpha-smooth muscle actin, a differentiation marker of smooth muscle cells, is present in microfilamentous bundles of pericytes.

Abstract: alpha-Smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle cells (SMC) and present in high amounts in vascular SMC, was demonstrated in the cytoplasm of pericytes of various rat and human organs by means of immunocytochemistry at the electron microscopic level. In SMC and pericytes, alpha-sm actin was localized in microfilament bundles, strengthening the assumption that it is the functional isoform in these cell types and supporting the assumption that pericytes exert contractile functions.

Distribution of tau proteins in the normal human central and peripheral nervous system.

Abstract: In human brain, antibodies to tau proteins primarily label abnormal rather than normal structures. This might reflect altered immunoreactivity owing to post-mortem proteolysis, disease, or species differences. We addressed this issue by comparing the distribution of tau in bovine and human post-mortem nervous system tissues and in human neural cell lines, using new monoclonal antibodies (MAb) specific for phosphate-independent epitopes in bovine and human tau. In neocortex, hippocampus, and cerebellum, immunoreactive tau was widely expressed but segregated into the axon-neuropil domain of neurons. In spinal cord and peripheral nervous system, tau immunoreactivity was similarly segregated but less abundant. No immunoreactive tau was detected with our MAb in glial cells or in human neural cell lines that express neurofilament or glial filament proteins. Post-mortem delays in tissue denaturation of less than 24 hr did not affect the distribution of tau, but the method used to denature tissues did, i.e., microwave treatment preserved tau immunoreactivity more effectively than chemical fixatives such as Bouin's solution, and formalin-fixed tissue samples reacted poorly with our anti-tau MAb. We conclude that the distribution of tau proteins in human nervous system is similar to that described in perfusion-fixed experimental animals, and that visualization of normal immunoreactive tau in human tissues is critically dependent on the procedures used to denature post-mortem tissue samples. Furthermore, microenvironmental factors in different neuroanatomical sites may affect the regional expression of tau.

Histochemical characterization of neuronal NADPH-diaphorase

Abstract: We examined the properties of neuronal NADPH-diaphorase in sections of rat striatum, using histochemical procedures. NADPH-diaphorase histochemistry stained discrete populations of central neurons ...

Distribution of immunoreactive Na+,K+-ATPase in gerbil cochlea.

Abstract: The distribution of Na+,K+-ATPase was mapped in cochleas of mature gerbils with normal hearing, using a specific and sensitive immunocytochemical method. Na+,K+-ATPase was abundant in the basolateral plasma membrane of marginal cells in the stria vascularis. Considerable levels of enzyme were also associated with the surfaces of spiral ganglion neurons and their central and peripheral processes. An unexpected finding was the detection of high levels of immunoreactive Na+,K+-ATPase in three different populations of cells lying in the inferior portion of the spiral ligament and at the medial and lateral border of the scala vestibuli just superior to the attachment of Reissner's membrane. Cells in these areas shared the morphological characteristics of cells specialized for active transport but appeared to be nonpolarized, suggesting a uniform distribution of Na+,K+-ATPase over their entire plasmalemma. The presence of these three distinct cell populations in the cochlea of several mammalian species suggests that they play an important role in cochlear function, perhaps that of regulating the cation content of perilymph. The absence of discrete concentrations of Na+,K+-ATPase-rich cells in the perilymphatic connective tissue of the bird cochlea and the mammalian vestibular system suggests further that these cells may be involved with generating and maintaining the high endolymphatic potential unique to the mammalian cochlea.

Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice.

Abstract: SPARC (Secreted Protein that is Acidic and Rich in Cysteine), a Ca++-binding glycoprotein also known as osteonectin, is produced in significant amounts by injured or proliferating cells in vitro. To elucidate the possible function of SPARC in growth and remodeling, we examined its distribution in embryonic and adult murine tissues. Immunohistochemistry on adult mouse tissues revealed a preferential association of SPARC protein with epithelia exhibiting high rates of turnover (gut, skin, and glandular tissue). Fetal tissues containing high levels of SPARC included heart, thymus, lung, and gut. In the 14-18-day developing fetus, SPARC expression was particularly enhanced in areas undergoing chondrogenesis, osteogenesis, and somitogenesis, whereas 10-day embryos exhibited selective staining for this protein in Reichert's membrane, maternal sinuses, and trophoblastic giant cells. SPARC displayed a Ca++-dependent affinity for hydrophobic surfaces and was not incorporated into the extracellular matrix produced by cells in vitro. We propose that in some tissues SPARC associates with cell surfaces to facilitate proliferation during embryonic morphogenesis and normal cell turnover in the adult.

Tissue distribution of antileukoprotease and lysozyme in humans.

Abstract: We studied the distribution of the proteinase inhibitor antileukoprotease (ALP) and lysozyme (LSZ) in normal human tissues by use of an immunohistochemical technique. ALP could be demonstrated in a variety of healthy tissues, generated by secretory cells in lacrimal, respiratory, proximal digestive, and genital glands, but not in the urinary tract, endocrine glands, or the hematological system. In lung, part of the non-ciliated cell population in membranous bronchiolar epithelium, as well as in respiratory bronchioles, stained positively for the inhibitor. The distribution of ALP and LSZ was parallel in most but not all of the tissues examined. The distribution of ALP around most of the external orifices of the human body is discussed in relation to its possible local physiological role in defense against inflammatory reactions. The widespread parallel distribution of LSZ, a bacteriolytic agent, suggests a complementary role to ALP in local defense.

Two simple methods for quantifying low-affinity dye-substrate binding.:

Abstract: Binding with low-affinity ligands, such as histological dyes, can be difficult to quantitate owing to the dissociation of bound ligand with washing or the retention of nonspecifically bound ligand because of incomplete washing. The present report describes two simple, rapid methods of discriminating bound from free ligand without the need for washing steps. One method is based on the spectral changes induced in a dye ligand, Congo red, on binding to the "receptor" insulin fibrils. This method discriminates spectrophotometrically between bound and free ligand without requiring any physical separation of the two forms. No radioactive ligands are necessary, and, by using disposable cuvettes, the entire binding assay can be done in a single container without the need for transfers. The second method employs a non-traditional filtration approach that avoids the need for a washing step by measuring the decrease in concentration of the dye ligand in the filtrate rather than by applying the usual approach of meas...

Effects of prolonged ethanol exposure on the glial fibrillary acidic protein-containing intermediate filaments of astrocytes in primary culture: a quantitative immunofluorescence and immunogold electron microscopic study.

Abstract: We investigated the effects of ethanol exposure on the shape of the cell and the morphology of intermediate filaments (IF) of cortical astrocytes in primary culture. The content and distribution of glial fibrillary acidic protein (GFAP), the major component of glial IF, was assessed using an anti-GFAP monoclonal antibody and fluorescence scanning densitometry together with quantitative pre- and post-embedding immunogold electron microscopy. The astrocytes were from 21-day-old fetuses obtained from both control and chronic alcoholic rats and were cultured for 28 days in the absence or presence of ethanol (25 mM). The main findings were: (a) ethanol-exposed astrocytes failed to develop processes or to acquire a filamentous IF distribution pattern; (b) these cells showed less GFAP than astrocytes without alcohol; (c) ethanol interfered with the reorganization of the anti-GFAP binding sites from clustered to random; and (d) astrocytes from alcohol-exposed fetuses cultured in the absence of ethanol also showed these alterations, suggesting initial damage to astrocyte precursor cells. Since the glial filaments play a crucial role in creating a scaffolding that guides neuronal migration, the effect of ethanol on astrocyte IF may possibly be correlated with the mechanisms underlying mental retardation and motor dysfunction which are characteristics of fetal alcohol syndrome.

Ecto-5'-nucleotidase: localization in rat kidney by light microscopic histochemical and immunohistochemical methods.:

Abstract: We demonstrated the distribution pattern of ecto-5'-nucleotidase (5'-Nu) in rat kidney by enzymatic activity (lead salt precipitation) and by immunohistochemistry with a polyclonal antibody raised in rabbits. Enzyme activity was found in the brush border of the proximal tubule, highest in the P1 segments with decreasing intensity in the P2 segments and weakest in P3 segments in the medullary rays of the cortex. The P3 segments of the outer stripe showed slightly higher activity. Activity was also apparent in the intercalated cells in the connecting tubule and collecting duct, whereas all other tubular and glomerular structures were negative. Activity in peritubular and perivascular connective tissue was highest in the cortical labyrinth, weak or absent in the medullary rays of the cortex, and entirely absent in the medulla. The distribution of the antigen was fully congruent with that of the enzyme activity. With respect to the role of adenosine in regulation of renal blood flow and glomerular filtration rate, the distribution of 5'-Nu in the cortical interstitium may be particularly significant. The possibility of nucleotide cleavage at the brush-border membranes may be important for salvage of nucleotides from the tubular lumen.

Expression of the neural cell adhesion molecule NCAM in endocrine cells.

Abstract: We examined the expression of the neural cell adhesion molecule NCAM in a number of endocrine tissues of adult rat and in an endocrine tumor cell line. NCAM was found by immunoelectron microscopy to be present on the surface of all endocrine cells in the three lobes of the hypophysis, although staining was relatively less intense in the intermediate lobe, and in pancreatic islets. Pituicytes, hypophyseal glial cells, were also labeled for NCAM. A rat insulinoma cell line (RIN A2) also expressed NCAM as judged by immunocytochemistry. Analysis of NCAM antigenic determinants (Mr 180, 140, and 120 KD) revealed large variations in the relative proportions of NCAM polypeptides present in the different tissues. Although all tissues and cell lines expressed NCAM-140, NCAM-180 was not detected in the adenohypophysis, pancreas, or adrenal medulla, and NCAM-120 was found in none of the endocrine tissues or cell lines except at low levels in the neurohypophysis. The tumor cell line expressed significant levels of NCAM-180, which was most abundant in the neurohypophysis. These results show that NCAM expression appears to be a general property of endocrine cells, although the antigenic composition differs markedly from that in brain tissue. These data are discussed with regard to the embryological origins of the different endocrine tissues, and possible functional implications are suggested.

Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus.

Abstract: The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, ie, the dense fibrillar component and the granular component Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers

Binding and uptake of pulmonary surfactant protein (SP-A) by pulmonary type II epithelial cells.

Abstract: A glycoprotein of Mr 26-36,000 (SP-A) is an abundant phospholipid-associated protein in pulmonary surfactant. SP-A enhances phospholipid reuptake and inhibits secretion by Type II epithelial cells in vitro. We have used two electron microscopic cytochemical methods to demonstrate selective binding and uptake of SP-A by rat pulmonary Type II epithelial cells. Using an immunogold bridging technique, we showed that SP-A binding was selective for Type II cell surfaces. Binding was dose dependent and saturable, reaching maximal binding at approximately 10 ng/ml. On warming to 23 degrees C, SP-A binding sites were clustered in coated pits on the cell surface. To characterize the internalization and intracellular routing of SP-A, we used the biotinyl ligand-avidin-gold technique. Biotinyl SP-A was bound by rat Type II epithelial cells as described above. On warming, biotinyl SP-A was seen in association with coated vesicles and was subsequently located in endosomes and multivesicular bodies. Biotinyl SP-A-gold complexes were seen in close approximation to lamellar bodies 10-60 min after warming. Binding of biotinyl SP-A was inhibited by competition with unlabeled SP-A. These results support the concept that Type II epithelial cells bind and internalize SP-A by receptor-mediated endocytosis. This newly described uptake system may play a role in the recycling of surfactant components or mediate the actions of SP-A on surfactant phospholipid secretion.

Ultrastructural localization of neuropeptides and GABA in rat dorsal horn: a comparison of different immunogold labeling techniques.:

Abstract: Several immunogold techniques were used to determine the ultrastructural localization of calcitonin gene-related peptide (CGRP), tachykinin, somatostatin, and gamma-amino-butyric acid (GABA) immunoreactivity in the dorsal horn of rat spinal cord. The immunocytochemical reactions were carried out directly on ultrathin sections from non-osmicated frozen tissue, non-osmicated low temperature-embedded (Lowicryl K4M) tissue, and osmicated epoxy-embedded material. Preservation of ultrastructural morphology and immuno-labeling efficiency were compared. Morphology of subcellular organelles was relatively good in ultra-thin frozen sections, which showed the highest immunoreactivity. However, only very small samples of tissue could be examined. Although there was relatively good immunolabeling in the Lowicryl K4M-embedded tissue, the ultrastructure of the neuropil, and particularly that of synapses, was poorly maintained. In contrast, the osmicated epoxy-embedded material offered optimal morphological preservation ...

Double immunocytochemical labeling of cell and tissue samples with monoclonal anti-bromodeoxyuridine.

Abstract: We describe a new monoclonal antibody (designated Bu20a) against bromodeoxyuridine (BrdU). This antibody was selected by screening against human tissues using the APAAP technique, and shows no crossreactivity with normal nuclei. It stains BrdU incorporated into the nuclei of a wide range of cell types, including human tonsil lymphoid cells, normal mouse tissues, and human tumors growing in nude mice. A double-labeling technique is described using this antibody in which cell smears or tissue sections are first labeled by an immunoperoxidase procedure for a cellular antigen (e.g., mouse or human histocompatibility class II antigen, T-lymphocyte antigen, keratin) and BrdU is then detected by indirect immunofluorescence. This procedure, which was applied to a variety of human and animal cells and tissues, is of wide potential value in analyzing the phenotype of S-phase cells and in co-localizing antigen expression and BrdU incorporation in tissue sections.

A highly sensitive one-step method for silver intensification of the nickel-diaminobenzidine endproduct of peroxidase reaction.

Abstract: We have developed a new technique which makes silver intensification of the oxidatively polymerized diaminobenzidine (DAB), the endproduct of peroxidase reaction, less laborious without any loss in selectivity or sensitivity. The new technique is based on two strategies: (a) increasing the argyrophilia of the DAB by modifying its polymerization with Ni ions, and (b) decreasing tissue argyrophila by using a mildly acidic physical developer instead of the alkaline one previously presented. Because the nickel modification takes place in the DAB substrate solution, i.e., in the final step of the peroxidase reaction, only one additional step, the physical development, must be carried out if intensification is needed.

Acute action of DSP-4 on central norepinephrine axons: biochemical and immunohistochemical evidence for differential effects.

Abstract: Previous immunohistochemical studies of the long-term effects of the noradrenergic neurotoxin DSP-4 have demonstrated a remarkably selective vulnerability of norepinephrine (NE) axons of the locus coeruleus (LC). NE axons originating in non-LC NE neurons appear to be largely resistant to the neurotoxic action of DSP-4. We conducted this study to evaluate the acute effects of DSP-4 on NE axons in four different brain regions: cerebral cortex, cerebellum, ventral forebrain, and hypothalamus. NE levels were determined by high-performance liquid chromatography (HPLC) 6 and 24 hr and 14 days after DSP-4 administration. NE axons in these brain regions were visualized in brain sections at 6 and 24 hr after drug treatment, using a specific antiserum to NE. HPLC assays revealed profound reductions of NE levels in cerebral cortex and cerebellum, but only minor decreases in ventral forebrain and hypothalamus. NE immunohistochemistry showed dramatic differences in the acute effects of DSP-4 on NE axon staining: nearly complete loss of staining in cortex and cerebellum, in contrast to an almost unchanged staining pattern in ventral forebrain and hypothalamus. This study demonstrates that NE immunohistochemistry is a valuable tool to assess the acute effects of DSP-4 on NE axons in different brain regions. The results provide the first direct evidence that NE axons are not uniformly acted on by DSP-4 and suggest that the acute effects of DSP-4 are restricted to LC axons.

Immunohistochemical localization of metallothionein in developing rat tissues

Abstract: Metallothionein (MT) is a cysteine-rich, low molecular weight protein inducible by heavy metal ions and various endogenous factors. Using an indirect immunofluorescent technique, we studied the localization of MT in developing rat tissues (kidney, small intestine, and liver). In kidney of the neonate and fetus, MT was found in both the cytoplasm and the nucleus of renal tubular epithelia. Localization of MT changed with shift of zonation in the renal cortex during development. Metallothionein was found mainly in the inner zone of the cortex but not in tubules of the neogenic zone on Day 4. Until Day 18, tubular cells containing MT were observed in a part of the cortex adjacent to the medulla, followed by a significant decrease in immunostaining by Day 27. In small intestine of the neonate, MT was localized predominantly in Paneth and goblet cells which play secretory roles. The number of goblet cells with strong immunostaining for MT was maximal on Day 27. In liver of 20-day fetuses and of 4-day-old neona...

Double labeling with iodo- and bromodeoxyuridine for cell kinetics studies.

Abstract: The rate of progression through the cell cycle was determined in five human glioma cell lines by a new sequential immunohistochemical staining technique. The cells were labeled first with iododeoxyuridine (IdUrd) for 1-3 hr and then with bromodeoxyuridine (BrdUrd) for 30 min. Labeled cells were identified with Br-3, a monoclonal antibody that recognizes only BrdUrd, and with IU-4, an antibody that recognizes both IdUrd and BrdUrd. Each slide was stained sequentially, first with the immunoperoxidase method for Br-3 and then with the alkaline phosphatase-anti-alkaline phosphatase method for IU-4. Cells that were positive only for IU-4 represented the fraction of S-phase cells that passed into the G2 phase during the period of incubation with IdUrd. The rates of progression measured by this method were constant in each cell line and resulted in smaller standard errors than were obtained by measurements from specimens stained singly for IdUrd and BrdUrd in different slides. The duration of the S-phase calculated from this fraction in the five cell lines ranged from 8-13 hr; the estimated potential doubling times were 25-32 hr and were very similar to the actual doubling times.

Heterogenous distribution of type IV collagen, entactin, heparan sulfate proteoglycan, and laminin among renal basement membranes as demonstrated by quantitative immunocytochemistry.

Abstract: Type IV collagen, entactin, heparan sulfate proteoglycan, and laminin antigenic sites were revealed on various rat renal basement membranes by use of protein A-gold immunocytochemistry. The basement membranes of the proximal and distal convoluted tubules, those of Bowman's capsule and glomerulus, and the mesangial matrix were labeled for all the antigens but to differing extents. Control experiments confirmed the specificity of these labelings. Quantitative evaluation revealed an important heterogeneity for each antigen among the various basement membranes. This heterogeneity suggests that the basement membrane components must arrange themselves in different ways, possibly to account for differences in functional properties of the various renal structures.

Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic islets.

Abstract: We examined transthyretin immunoreactivity (TTR-IR) in human and porcine liver, choroid plexus, and pancreatic islets with both polyclonal and monoclonal antibodies to TTR. The specificity of the immunoreactions and the effects of various fixatives were tested in immunohistochemical and dot-blot systems. B-5 fixative (mercuric chloride and sodium acetate in formalin) was the best immunopreservative. In both species, the TTR-IR in choroid plexus epithelial cells was strong and was much greater than that in hepatocytes. Glucagon cells in pancreatic islets were also strongly TTR immunoreactive. Insulin cells were slightly TTR immunoreactive in human but strongly so in porcine pancreas. The finding of TTR-IR in normal islets explains the presence of TTR-IR in human endocrine pancreatic tumors, notably glucagonomas and malignant insulinomas.

A double-label pre-embedding immunoperoxidase technique for electron microscopy using diaminobenzidine and tetramethylbenzidine as markers.

Abstract: Techniques for correlative double-label immunocytochemistry (ICC) at light and electron microscopic (EM) level are useful for determining the neurotransmitter phenotype of inputs onto immunocytochemically identified neurons. Tetramethylbenzidine (TMB) has been used as a chromogen at the EM level for horseradish peroxidase tract tracing. We have found that TMB, in combination with diaminobenzidine (DAB), can be used in a double-label immunocytochemical protocol to examine neuropeptide Y inputs onto luteinizing hormone-releasing hormone cells in the sheep preoptic area. At both light and EM levels, TMB reaction product is visibly distinct from DAB reaction product. The ultrastructural preservation we have been able to obtain with our technique is better than that obtained with techniques that use TMB at a lower pH. Furthermore, this technique allows the demonstration of synaptic contacts between neurochemically identified terminals and cells with different neurotransmitter phenotypes.

Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling.

Abstract: We labeled active S-phase cells in primary breast carcinomas with a modified 5-bromo-2'-deoxyuridine (BrdU) procedure using a silver-enhanced colloidal gold visualization step. Separate samples of ...

Tartrate-resistant acid ATPase as a cytochemical marker for osteoclasts.

Abstract: We present a modified histochemical method for staining osteoclasts and adjacent mononuclear cells which takes advantage of the recently described substrate specificity for ATP of osteoclastic acid phosphatase. Staining of osteoclasts using ATP as substrate exhibits by light microscopy the same tartrate resistance as conventional acidic phosphatases, without the bone surface staining seen with other substrates. This feature, coupled with specific staining of fewer vicinal mononuclear cells, makes this method potentially useful for studying osteoclast ontogeny and function.

Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct.

Abstract: Ultrastructural localization of the alpha-subunit of Na+,K+-ATPase on the lateral wall of rat cochlear duct was investigated quantitatively by the protein A-gold method, using affinity-purified ant...

Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection

Abstract: To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.

Synthesis of a more stable osmium ammine electron-dense DNA stain.

Abstract: Specific DNA staining for electron microscopic observation is simplified by a shorter synthesis of the staining reagent. The new, more reliable reagent, osmium ammine-B, is stable for more than a year, dissolves completely in water, and does not require reoptimization of staining conditions for every batch, yet reproducibly gives strong contrast to DNA-containing structures.

Immunocytochemical Localization of Basic Fibroblast Growth Factor in Bovine Adrenal Gland, Ovary, and Pituitary'

Abstract: We studied the distribution of basic fibroblast growth factor (bFGF) immunoreactivity in bovine adrenal gland, ovary, and pituitary, using a polyclonal anti-bFGF antibody. In the adrenal gland, the inner layers of the capsule, the zona glomerulosa of the cortex, and the chromaffin cells of the adrenal medulla were intensely stained. In the ovary, follicular epithelial cells of growing follicles and granulosa cells of mature follicles showed strong bFGF-like immunoreactivity. Endocrine cells of the pituitary anterior and intermediate lobes displayed a positive immunoreaction. Blood vessels, including endothelial and smooth muscle cells, as well as stromal cells in all three organs studied, were not stained. This distribution pattern of bFGF immunoreactivity is only partially compatible with the established mitogenic role of this protein, and suggests a wider spectrum of bFGF functions.