bluuebunny/crossref_metadata_2025 huggingface dataset:
Production and characterization of monoclonal antibody to dermatan sulfate proteoglycan.
Authors: Sobue M , Nakashima N , Fukatsu T , Nagasaka T , Katoh T , Ogura T , Takeuchi J
Published: April 2011

We purified dermatan sulfate proteoglycan (PG) from the capsule of human ovarian fibroma for use as an immunogen. A monoclonal antibody, designated 6B6, was produced which reacts to the intact molecule of dermatan sulfate PG and the chondroitinase AC-treated core molecule on Western-blotted nitrocellulose membrane. Localization of materials showing crossreactivity to this antibody was studied in human tissues by indirect immunohistochemistry. The interstitial elements of almost all tissues examined were positive for the antibody: dermis, submucosal layer of digestive tract, perichondral layer, perivascular connective tissue, perineurium, adventitia of aorta, vessel wall of vein, pleura, and fibrous capsule of kidney and liver. Positive staining was also observed in fibrous elements at post-necrotic foci of cardiac muscle and pancreas, and at atherosclerotic lesions of aorta. The distribution of the antigen, core protein of the dermatan sulfate PG, revealed with 6B6 was compared to that of the dermatan sulfate side chain, which was demonstrated with antibody 9A-2 (Couchman et al.: Nature 307:650, 1984) after treatment with chondroitin sulfate B-lyase. The distribution of both antigens, core protein, and dermatan sulfate side chains showed the same pattern, with minor exceptions. The antibody 6B6 will be a useful tool to study the immunohistochemical localization of dermatan sulfate PG.
Photoconversion of some fluorescent markers to a diaminobenzidine product.
Authors: Sandell J H , Masland R H
Published: April 2011

Retinal whole mounts, brain sections, and astrocyte cultures were labeled with various fluorescent markers. Tissues or cells were then irradiated by light in the presence of diaminobenzidine. Irradiation initiated a reaction in which specific fluorescent labeling was replaced by an insoluble diaminobenzidine product. The diaminobenzidine product is more stable than the original fluorescent labeling and can be processed for electron microscopy. In some cases, the reaction product reveals cellular detail that cannot be resolved in the fluorescent labeling. The 10 fluorescent markers tested have widely differing structures, span a broad range of wavelengths, and label several different cellular elements. The photoconversion reaction was successful with all markers and tissues tested.
Calcitonin and chromogranin A localization in medullary carcinoma of the thyroid by immunoelectron microscopy.
Authors: Silver M M , Hearn S A , Lines L D , Troster M
Published: April 2011

We used a post-embedding immunoelectron microscopy method, using protein A-gold, to detect calcitonin and chromogranin A immunoreactivity in three cases of human medullary thyroid carcinoma. Because the epoxy-embedded tissue had been fixed (glutaraldehyde or formaldehyde) and osmicated before embedment, the proteins were identified in optimally preserved tissue. Uranyl and lead staining was used after immunolabeling, so that the tissue was also optimally contrasted. The morphological advantage provided by osmication was tested by labeling rat thyroid gland C-cells for calcitonin. The protein A-gold technique allowed localization of both antigens to the contents of membrane-bound secretory granules in the tumor cells. In one case, labeling density for each antigen was measured over several intercellular compartments and the interstitium. Calcitonin, but not chromogranin A, reactivity was also identified in intracellular amyloid fibrils in two cases, showing that the constant region of calcitonin is preserved in amyloid deposits related to the tumor cells.
Effects of acute doses of sodium fluoride on the morphology and the detectable calcium associated with secretory ameloblasts in rat incisors.
Authors: Monsour P A , Harbrow D J , Warshawsky H
Published: April 2011

Fluoride in high concentrations is known to have an adverse effect on the formation of enamel. The effect of a single injection of two concentrations of sodium fluoride on inner enamel secretory ameloblasts was investigated morphologically by electron microscopy and functionally by assessing the location and relative amount of available calcium, using the potassium pyroantimonate method. The results showed that acute doses of fluoride interfere with the normal function of secretory ameloblasts. The increase in the population of lysosome-like structures observed after fluoride administration is suggestive of defects in the synthetic pathway. Concomitant with the effect of fluoride on secretory ameloblasts is an inhibition of enamel formation, resulting in incomplete enamel rods and leaving large remnants of Tomes' processes buried in the enamel. The distribution of the calcium pyroantimonate deposits found tends to support the concept of calcium traveling between the cells to the enamel. Acute doses of fluoride also reduce the amount of calcium available for complexing with pyroantimonate in the intercellular region.
Autoradiographic visualization of alpha 1-adrenergic receptors in cervix of early pregnant rat.
Authors: Maltier J P , Petit I , Legrand C
Published: April 2011

alpha 1-Adrenergic receptors were identified, characterized, and localized in rat cervix on Day 6 of pregnancy by autoradiography. Autoradiographic study was performed in slide-mounted rat cervix sections using [3H]-prazosin ([3H]-PRAZ) as ligand. Binding was time dependent and specific. Pharmacological study indicated that specific [3H]-PRAZ binding was inhibited with high affinity by prazosin and phenylephrine and low affinity by yohimbine and clonidine. In cervix, the alpha 1-adrenergic receptors were localized mainly to the inner circular layer of the myometrium. Binding to the outer longitudinal layer of myometrium was moderate, and binding was absent in the endometrium. The regional distribution of alpha 1-adrenergic receptors strongly suggests that the circular layer of myometrium may function as an important modulator of contractile response of the cervix, probably involved in the retention of blastocysts at the utero-cervical end of the horn.
Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons.
Authors: Chalmers G R , Edgerton V R
Published: April 2011

The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.
Glycoconjugates on corneal epithelial surface: effect of neuraminidase treatment.
Authors: Hazlett L D , Mathieu P
Published: April 2011

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.
Quantitative histochemical analysis of glucose-6-phosphatase activity in rat liver using an optimized cerium-diaminobenzidine method.
Authors: Jonges G N , Van Noorden C J , Gossrau R
Published: April 2011

We have optimized a cerium-diaminobenzidine-based method for histochemical analysis of glucose-6-phosphatase (G6Pase) activity and have determined quantitative data on the zonal distribution pattern in the liver acinus of fasted male rats. In the cerium-diaminobenzidine technique, cerium instead of lead ions is used as capturing reagent for the enzymatically liberated phosphate. For light microscopy, the primary reaction product, cerium phosphate, is then visualized by conversion into cerium perhydroxide using hydrogen peroxide and subsequent oxidative polymerization of diaminobenzidine to diaminobenzidine brown as the final reaction product. Variation of the substrate (glucose-6-phosphate) concentration in the incubation medium yielded in periportal zones a KM value of 2.3 +/- 0.7 mM and a Vmax value of 0.96 +/- 0.18 (expressed as mean integrated absorbance). In perivenous zones a KM value of 1.1 +/- 0.4 mM and a Vmax value of 0.51 +/- 0.08 were calculated. The cytophotometric analysis performed in this study demonstrated for the first time that a functional difference of G6Pase, the key enzyme for gluconeogenesis, exists in the periportal and perivenous zones of the liver acinus. Periportal zones contain twice as many enzyme molecules (high Vmax) as perivenous zones, but the affinity for the substrate is twice as low. This may have important implications for the concept of metabolic zonation of the liver and also for glucose homeostasis in the blood.
Immunocytochemical localization of the 34 KD pregnenolone-binding protein to fasciculata and reticularis cells and a novel 32 KD protein specific for reticularis cells in guinea pig adrenal cortex.
Authors: Whitnall M H , Lee Y C , Driscoll W J , Strott C A
Published: April 2011

Two proteins were isolated and purified from guinea pig adrenal cortex: a 34 KD protein that specifically binds pregnenolone (product of the rate-limiting step in steroidogenesis), and a novel co-purifying 32 KD protein that has not been characterized. Specific antisera were generated and used for immunocytochemical analysis. The 34 KD and 32 KD proteins were specific for the adrenal cortex and were absent from other tissues, including the testis. The 34 KD pregnenolone binding protein (PBP) was localized to zona fasciculata and zona reticularis cells and absent from zona glomerulosa cells. Thus, the PBP was absolutely correlated with ACTH-regulated steroidogenic cells, whereas steroidogenic cells regulated by other peptide hormones did not contain the PBP. This finding suggests a functional relationship between the PBP and ACTH. A second interesting finding was that a novel 32 KD co-purifying protein localized to the zona reticularis and was absent from the zona glomerulosa and the zona fasciculata. The 32 KD protein can therefore serve as an excellent marker for the reticularis cell of the adrenal cortex.
Multimarker immunohistochemical staining of calgranulins, chloroacetate esterase, and S100 for simultaneous demonstration of inflammatory cells on paraffin sections.
Authors: Chilosi M , Mombello A , Montagna L , Benedetti A , Lestani M , Semenzato G , Menestrina F
Published: April 2011

Mac387 monoclonal antibody (MAb) recognizes two calcium binding, myeloid-associated proteins, now termed calgranulins, expressed at high levels by neutrophils and monocytes. Calgranulins are related to migration inhibitory factor (MIF) and are lost in a few days from monocytes differentiated in vitro. This marker is therefore potentially useful to analyze macrophage heterogeneity and turnover in tissue sections. In this study, we developed an immunohistochemical multimarker technique, including calgranulin demonstration, suitable for analyzing different inflammatory cells on paraffin-embedded material. The technique was carried out in subsequent steps demonstrating (a) naphthol AS-D chloroacetate esterase (CAE); (b) S100 immunoreactivity using a rabbit antibody in peroxidase-antiperoxidase (PAP) staining; and (c) Mac387 immunoreactivity using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. CAE staining was introduced in this method to distinguish Mac387+/CAE- macrophages from Mac387+/CAE+ neutrophils, and Mac387-/CAE+ mast cells. S100 protein is strongly expressed within lymphoid tissues by dendritic accessory cells and was then applied to distinguish these cells from S100-macrophages. We have also verified the possibility of reducing the staining time for this time-consuming procedure by use of microwave irradiation. The technique was applied to a representative variety of normal and pathological samples to assess its usefulness for study of cell heterogeneity. Our results showed the multimarker technique to be highly informative in the study of inflammatory lesions (e.g., rheumatoid arthritis, sarcoid and cat-scratch granulomas, dermathopathic lymphadenopathy), and is of wide potential value as an aid to histopathological diagnosis of several diseases.
Influence of age on NMDA receptor complex in rat brain studied by in vitro autoradiography.
Authors: Kito S , Miyoshi R , Nomoto T
Published: April 2011

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimer's disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine. Both [3H]-glycine and [3H]-CPP binding sites were most abundant in the hippocampus and cerebral cortex, and they showed a similar distribution pattern throughout the brain. [3H]-glycine binding sites were severely decreased in the telencephalic regions, including the hippocampus and cerebral cortex, in aged brain. Conversely, [3H]-CPP binding sites were well preserved in these brain areas. In the mid-brain regions and cerebellum, neither [3H]-glycine nor [3H]-CPP binding sites changed in the aged brain. Our results indicate that within the NMDA receptor complex, glycine receptors are primarily affected in the aging process.
BrdUrd labeling of S-phase cells in testes and small intestine of mice, using microwave irradiation for immunogold-silver staining: an immunocytochemical study.
Authors: Thoolen B
Published: April 2011

In this study, BrdUrd labeling of S-phase cells in the small intestine and testes was accomplished using microwave irradiation. In this way crypt cells, spermatogonia, and Leydig cells could be labeled using removable plastic-embedded sections and immunogold-silver staining (IGSS). By using short periods of microwave irradiation for incubation of the monoclonal antibodies and the protein A-colloidal gold solution, the detection of BrdUrd-labeled cells could be remarkably enhanced. A comparative study of BrdUrd labeled spermatogonia in the testis of a Cpb-N mouse that received both [3H]-thymidine and BrdUrd proved that 90% of the BrdUrd-labeled cells also showed [3H]-thymidine labeling. The radioactive [3H]-thymidine labeling was a time-consuming method of 4 weeks' duration, whereas the BrdUrd-labeled cells could be labeled, fixed, enhanced, and counterstained in less than 3 hr. This investigation proves that BrdUrd labeling of S-phase cells can be a reliable, reproductive, rapid, and non-radioactive alternative method for [3H]-thymidine labeling of proliferating cells.
Histochemical localization and analysis of blood group-related antigens in human pancreas using immunostaining with monoclonal antibodies and exoglycosidase digestion.
Authors: Ito N , Nishi K , Nakajima M , Okamura Y , Hirota T
Published: April 2011

We examined the distribution of blood group-related antigens using an indirect immunoperoxidase method with monoclonal antibodies (MAb) directed to A, B, H, Lewis a (Lea), Lewis b (Leb), Lewis x (Lex), and Lewis y (Ley) antigens and Type 1 precursor chain in human pancreas. Effects of prior digestion with exoglycosidases on MAb stainings were simultaneously investigated. A, B, H, Leb, and Ley antigens were detected in acinar cells and interlobular duct cells but not in centroacinar cells, intercalated duct cells, and islet of Langerhans cells. The expression of these antigens in acinar cells was not dependent on Lewis type and secretor status of the tissue donors, whereas that in interlobular duct cells was strictly dependent on secretor status. The distribution pattern of these antigens in acinar cells was not homogeneous, i.e., cells producing H antigens expressed both Leb and Ley antigens but not A or B antigens, whereas those producing A or B antigens did not secrete Leb and Ley as well as H antigens. Digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase resulted in the appearance of Leb and Ley antigens as well as H antigen in acinar cells producing A and/or B antigens. Type 1 precursor chain was not detected in pancreatic tissues from secretors but appeared in acinar cells producing H antigen after alpha-L-fucosidase digestion, which also disclosed Lex but not Lea antigen in acinar cells expressing both Leb and Ley. In some non-secretors, MAb against Type 1 precursor chain reacted with acinar cells without enzyme digestion. Although Lea antigen was not detected in acinar cells, it was found in centroacinar cells, intercalated duct cells, and interlobular duct cells from all individuals examined except two Le(a-b-) secretors. After sialidase digestion, Lex antigen appeared in centroacinar and intercalated duct cells from some individuals. Sialidase digestion also elicited reactivity with MAb against Type 1 precursor chain in islet of Langerhans cells from some individuals. These results demonstrate the complexity in the pattern of expression and regulation of blood group-related antigens in different cell types of human pancreas. Such complexity may largely be ascribed to differences in individual genotypes and in gene expression patterns of different cell types.
An electrophoretic method for selection of conditions for production of electrophoretically uniform protein colloidal gold complexes.
Authors: Geoghegan W D
Published: April 2011

An electrophoretic method was developed to determine the conditions required for production of electrophoretically uniform protein-colloidal gold complexes from monodisperse colloidal gold (Au) and electrophoretically uniform protein. The method is based on the electrophoretic migration of protein-Au complexes in agarose. The results demonstrate that two variables, the pH of adsorption and the quantity of protein added, can be manipulated to vary the electrophoretic mobility of the resulting protein-Au complexes. Thus, agarose gel electrophoresis can be used to select the pH of adsorption and the quantity of protein required to produce electrophoretically uniform protein-Au complexes. This new electrophoretic mobility test can be used in place of or in addition to the classical procedure of Zsigmondy and its many variations, both visual and spectrophotometric. The procedure described is also useful for electrophoretic comparison of small quantities of various protein-Au samples.
Dynamics of Ca2+ transients in norepinephrine-stimulated individual H-35 hepatoma cells: fura-2 digital imaging microscopy and high time-resolution microspectrofluorometry.
Authors: Yodozawa S , Tsunoda Y , Funai T , Tashiro Y
Published: April 2011

We investigated spatiotemporal changes in cytoplasmic free Ca2+ concentration ([Ca2+]i) in norepinephrine (NE)-stimulated and fura-2-loaded individual H-35 rat hepatoma cells, using digital imaging microscopy and high time-resolution microspectrofluorometry. Application of NE (5 x 10(-6) M) resulted in an initial transient increase in [Ca2+]i, followed by a small sustained [Ca2+]i plateau above the pre-stimulation level. The initial peak and the small sustained plateau originated from intracellular stores and the extracellular space, respectively. The initial transient evoked by NE was totally blocked by phentolamine, an alpha-adrenergic antagonist, but was not blocked by either pre-incubation with nominally Ca(2+)-free medium or by pre-treatment of cells with La3+. On the other hand, the sustained plateau was eliminated by Ca(2+)-free medium or La3+. Therefore, H-35 cells have a Ca(2+)-signaling pathway which is activated via alpha-adrenergic receptors. Mn2+ entered the cytosol after NE stimulation, as shown by quenching of fura-2. This indicates that H-35 hepatoma cells possess Mn(2+)-permeable Ca2+ channels at the plasma membrane. In addition, the Ca2+ efflux pattern from H-35 cells to the extracellular space during NE stimulation was visualized by digital imaging microscopy when free fura-2 was equilibrated between the cells and the extracellular space. The efflux of Ca2+ from H-35 begins between the initial [Ca2+]i transient and the sustained [Ca2+]i plateau.
Lectin histochemistry of mouse vagina during the estrous cycle.
Authors: Vrcić H , Horvat B , Damjanov I
Published: April 2011

Estrous cycle-related histochemical changes in the vaginal epithelium of sexually mature female mice were studied with 30 fluorescein isothiocyanate (FITC)-labeled lectins. On the basis of the staining pattern the lectins were divided into five groups: I, seventeen lectins that reacted with mucinous surface layer of proestrus. This group comprised two subgroups: Ia, seven lectins that reacted exclusively with the mucinous layer, and Ib, ten lectins that reacted with mucinous cells and the underlying squamous epithelium of proestrus; II, two lectins that reacted with squamous epithelium of proestrus only but were unreactive with mucinous cells; III, three lectins that reacted in a phase-specific manner with squamous epithelium; IV, six lectins that showed increased luminal surface reactivity in diestrus and/or metestrus; and V, eleven lectins that were unreactive with vaginal epithelium. These data indicate that the cyclic changes in the morphology of the vaginal epithelium are accompanied by distinct lectin reactivity patterns.
Activity of alkaline phosphatase in rat skeletal muscle localized along the sarcolemma and endothelial cell membranes.
Authors: Safadi A , Livne E , Silbermann M , Reznick A Z
Published: April 2011

Alkaline phosphatase (AP), a membrane-associated glycoprotein which enhances the hydrolysis of monophosphate esters at alkaline pH, is widely distributed in animal tissues. AP activity is increased in a variety of muscle disorders, i.e., myopathies and denervation. Established histochemical methods at the light microscopy level failed to demonstrate AP in skeletal muscles. In the present study we applied the Gomori lead nitrate method for ultrastructural examination of AP in rat gastrocnemius muscles and showed that the enzyme was linked to the sarcolemma of the striated muscle and to the membranes of endothelial cells in adjacent capillaries. In comparison with ATPase activity, AP activity was inhibited by both levamisole and a pH of 7.2, but not by ouabain. Hence, it appears that in skeletal muscles AP is active at a high pH and is bound to cell membranes.
Simultaneous immunocytochemical visualization of bromodeoxyuridine and neural tissue antigens.
Authors: Soriano E , Del Rio J A
Published: April 2011

5'-Bromodeoxyuridine (BrdU) is a thymidine analogue which can be detected by monoclonal antibodies (MAb). We have developed a method for the simultaneous visualization of BrdU and a wide range of neural antigens in paraformaldehyde-fixed brain sections. Pregnant mice were injected intraperitoneally with a single pulse of BrdU. Young adult offspring were processed for immunocytochemistry following a double immunoperoxidase sequence. BrdU was detected using diaminobenzidine (DAB) intensified with nickel ammonium sulfate and neural antigen-containing elements were visualized with DAB alone. BrdU-positive nuclei and tissue antigen-immunoreactive cells were easily differentiated. Furthermore, double-labeled cells characterized by the presence of a black immunoreactive nucleus surrounded by a brown immunopositive cytoplasm were unambiguously recognized. Satisfactory results were obtained using either MAb or polyclonal antibodies against a variety of cell antigens, including neuropeptides, CA++ binding proteins, and cytoskeletal components of the glial cells. The method reported here permits analysis of the neurogenesis and proliferation of subsets of neurons and glial cells, identified by immunocytochemical markers.
Synthesis of CDP-diacylglycerol by rat liver rough microsomes as visualized by electron microscopic autoradiography: relationship to GTP-stimulated membrane fusion.
Authors: Jolicoeur M , Kan F W , Paiement J
Published: April 2011

Using detergent-free conditions of incubation for the analysis of liponucleotide synthesis, we compared GTP-dependent formation of CDP-diacylglycerol (CDP-DG) and membrane fusion in RNA-depleted rough microsomes from rat liver. After incubation of stripped rough microsomes (SRM) in the presence of GTP and [5-3H]-CTP, radioactivity was recovered in lipid extracts and identified by thin-layer chromatography as a single spot which co-migrated with CDP-DG. The nucleotide requirement for CDP-DG synthesis and that for membrane fusion were observed to be identical. We next carried out an electron microscopic autoradiographic analysis on incubated membranes to determine the site of incorporation of [5-3H]-CTP. Silver grains were observed directly over the unilamellar membranes of natural vesicles. In confirmation of the biochemical data, quantitation of silver grain density indicated more grains over membranes incubated in the presence of GTP than over those incubated in the absence of this nucleotide. For membranes incubated in the presence of GTP, the grain density was similar over fused and unfused membranes in the same preparation. When SRM were incubated with the enzyme co-factors required for synthesis of phosphatidylinositol, a GTP-independent membrane fusion was observed by both transmission and freeze-fracture electron microscopy. Together with the biochemical and autoradiographic data, this suggests that phospholipid metabolism may be activated by GTP and lead to the fusion of RER membrane.
A simple method for coupling in situ hybridization and immunocytochemistry: application to the study of peptidergic neurons.
Authors: Bugnon C , Bahjaoui M , Fellmann D
Published: April 2011

We have devised a simple procedure for immunostaining of sections that have previously undergone autoradiographic visualization of mRNAs by in situ hybridization. Classical hybridocytochemistry techniques were performed first on cryostat sections of formaldehyde-fixed tissue. Standard methods were used for slide coating by emulsion dipping and for revelation, fixation, and coverslipping steps. The key to this method is the emulsion removal, or permeabilization, by a short trypsin incubation (0.2% for 20-30 sec) which facilitates the good access of antibodies used in a subsequent immunocytochemical technique to section epitopes. Usual immunofluorescence and immunoperoxidase procedures were successfully performed after this treatment. The immunoreactivity of several neuropeptides was well preserved after this procedure. In addition to its usefulness in our studies, this general method should be applicable to many other situations in which autoradiographic and immunocytochemical detections must be coupled.
Combination of in situ hybridization and immunocytochemistry to detect messenger RNAs in identified CNS neurons and glia in tissue culture.
Authors: Trimmer P A , Phillips L L , Steward O
Published: April 2011

We have developed a technique in which immunofluorescence is combined with in situ hybridization using cDNA and RNA probes to assess the expression and distribution of messenger RNAs (mRNA) by neurons and neuroglia in tissue cultures of the rat dentate gyrus. The probes used in this study include a cDNA probe for ribosomal RNA (rRNA) and an RNA probe (cRNA) for glial fibrillary acidic protein (GEAP), an intermediate filament protein subunit expressed by astrocytes in the central nervous system. Both ubiquitous (tubulin) and cell type-specific (MAP-2 and GEAP) antibodies were used to identify neurons and neuroglia in culture. Using this procedure, the mRNA for rRNA was found in the cell bodies and large processes of MAP-2-positive neurons and throughout the cytoplasm of GEAP-positive flat astrocytes. In process-bearing astrocytes, GEAP mRNA is concentrated in the cell body, although some hybridization also occurred in astrocyte cell processes. With this combined in situ hybridization-immunofluorescence technique, the expression and distribution of an mRNA can be examined in different immunocytochemically identified cell types under identical culture and hybridization conditions. It is also possible to determine if there is a differential subcellular distribution of an mRNA in a single cell and if the distribution of the mRNA reflects the distribution of the protein itself. Finally, this technique can be utilized to verify the specificity of probes for cell type-specific mRNAs and to determine appropriate hybridization conditions to produce a specific signal.
Antibodies to tubulin and actin bind to the surface of a human monocytic cell line, U937.
Authors: Por S B , Cooley M A , Breit S N , Penny R , French P W
Published: April 2011

Intermittent reports of cytoskeleton proteins (actin and tubulin) on the cell surface have appeared over the last 13 years. Whereas most have concentrated on lymphocytes, this study provides evidence for the presence of these proteins on the surface of a human cultured monocyte-like cell line, U937. Both actin and tubulin were detected on the surface of U937 cells by flow cytometry, using an indirect staining procedure based on biotin-streptavidin-phycoerythrin, chosen for greater sensitivity. By use of this procedure, the majority of viable unstimulated U937 cells stained positively for actin and tubulin, although the level of fluorescence intensity was low. With an antibody specific for tyrosine-tubulin, most of the surface tubulin was also found to be tyrosinylated. For vimentin, an intermediate filament protein abundantly present in the cytoplasm of U937 cells, no staining could be detected. Confirmation of the flow cytometry data for surface actin and tubulin on unstimulated U937 cells was achieved by direct vesualization using a confocal laser scanning microscope. When U937 cells were activated with PMA and LPS, a marked reduction in the level of cell surface actin and tubulin occurred. The role of cell surface actin and tubulin on unstimulated U937 cells, in terms of monocyte function, remains to be elucidated.
Identification of peroxidase-positive astrocytes by combined histochemical and immunolabeling techniques in situ and in cell culture.
Authors: Schipper H M , Mateescu-Cantuniari A
Published: April 2011

A subpopulation of astrocytes in the vertebrate brain and in cysteamine-treated brain cell cultures contain cytoplasmic granules which exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic endogenous peroxidase activity. Visualization of these cells at the light microscopic level is confounded by the nonspecificity of the various histochemical methods routinely employed. In an attempt to circumvent this problem, we assayed for peroxidase-positive astrocytes using various combinations of diaminobenzidine (DAB) histochemistry and immunolabeling for the astrocyte-specific marker glial fibrillary acidic protein (GFAP). We determined that (a) DAB histochemistry in conjunction with avidin-biotin-immunoperoxidase labeling for GFAP specifically detects peroxidase-positive astrocytes in situ and (b) DAB histochemistry combined with indirect immunofluorescence for GFAP effectively demonstrates these cells in cysteamine-treated brain cell cultures.
Renal tubule gp330 is a calcium binding receptor for endocytic uptake of protein.
Authors: Christensen E I , Gliemann J , Moestrup S K
Published: April 2011

gp330, a large glycoprotein located in renal proximal tubules, has sequence similarities with the low-density lipoprotein receptor and the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. The 40 KD human alpha 2-macroglobulin receptor-associated protein is a newly discovered heparin binding protein homologous to a major rat Heymann nephritis factor and exhibiting high affinity binding to the alpha 2-macroglobulin receptor. The present study shows by ligand blotting, light and electron microscopic autoradiography, and cytochemistry that gp330 located in coated apical membrane regions of the rat proximal tubule strongly binds the 40 KD protein. Furthermore, 45Ca2+ blotting experiments disclosed gp330 as a quantitatively important Ca2+ binding protein in renal cortex. Binding of 125I-labeled 40 KD protein to electroblotted gp330 and to coated apical membrane regions in sections of renal proximal tubules was abolished by excess unlabeled 40 KD protein, heparin, and EDTA. The endocytic properties of gp330 were investigated by in vivo microperfusion of rat proximal tubules. After 6 min, 125I-labeled 40 KD protein was mainly found in endocytic vacuoles and later accumulated in lysosomes. These data demonstrate that gp330 is a Ca2+ binding receptor for endocytosis of protein and is functionally related to the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Furthermore, our results demonstrate the usefulness of semi-thin and ultra-thin cryosections in studies of ligand binding and subcellular localization of receptors with autoradiographic techniques.
Localization of heparan sulfate proteoglycan in basement membrane by side chain staining with cuprolinic blue as compared with core protein labeling with immunogold.
Authors: Chan F L , Inoue S , Leblond C P
Published: April 2011

We localized heparan sulfate proteoglycan (HSPG) in the basement membranes of ciliary epithelium and plantar epidermis, using Cuprolinic blue to stain its side chains and an immunogold procedure to detect its core protein. In accord with most of the literature, staining with Cuprolinic blue in glutaraldehyde fixative yielded three to five times as many reaction products along the two surfaces than along the center of the lamina densa, whereas immunogold labeling for the core protein after formaldehyde fixation yielded about twice as many gold particles over the center than along the surfaces of the lamina densa. It therefore appeared that HSPG side chains predominated outside, and the core protein within, the lamina densa. To find out whether the discrepancy was true or was an artifact caused by differences in processing, we attempted to combine the two approaches on the same material. This was found possible when Cuprolinic blue was used in formaldehyde fixative, embedding was in LR White, and immunogold labeling was performed on thin sections as usual. Under these conditions, both Cuprolinic blue reaction products and immunogold particles predominated over the lamina densa in the two basement membranes under study. Moreover, evidence was present that reaction products and immunogold particles either overlapped each other or were closely associated. The lens capsule (a thick basement membrane) also showed their co-localization. The discrepancy initially observed between side chains and core protein location was attributed to differences in processing, since Cuprolinic blue staining had been carried out in the course of glutaraldehyde fixation whereas immunogold labeling was done after formaldehyde fixation. The results lead to two conclusions. First, processing differences may alter the localization of HSPG and possibly other proteoglycans. Second, both HSPG side chains and core protein are localized in the same sites within basement membrane.
Distribution of a novel peptide in the anterior pituitary, gastric pyloric gland, and pancreatic islets of rat.
Authors: Yamamoto T , Katsumata N , Tachibana K , Friesen H G , Nagy J I
Published: April 2011

We used Western blot and immunohistochemical methods to investigate the biochemical characteristics and cellular distribution of a novel peptide (peptide 23) that was previously shown to be released from anterior pituitary cells of rat in response to growth hormone-releasing hormone. In the pituitary, peptide 23 isolated from intact cells had an Mr of 31,000, whereas that released into culture medium had an Mr of 16,000. Pancreatic islets contained a 19 KD form of the peptide. Immunohistochemically, peptide 23 in the rat pituitary gland was localized in a subpopulation of somatotropes. In pancreatic islets, the peptide was found by triple immunofluorescence labeling to be present in both insulin- and somatostatin-containing cells. In the gastrointestinal tract, peptide 23 was found only in a subpopulation of endocrine cells in the pyloric glands. This subpopulation of cells was found to be entirely separate from those containing either serotonin or somatostatin, and may represent one of the other known or as yet biochemically uncharacterized cell types in this gland. The results suggest that in response to secretagogues in vitro, an altered form of the peptide is secreted from pituitary cells and that an intracellular form of peptide 23 is expressed in some but not all somatotropes, a large proportion of islet cells, and a distinct population of pyloric cells.
Simultaneous fluorescence immunophenotyping and interphase cytogenetics: a contribution to the characterization of tumor cells.
Authors: Weber-Matthiesen K , Winkemann M , Müller-Hermelink A , Schlegelberger B , Grote W
Published: April 2011

In immunocytochemical studies, the phenotypic evaluation of tumor cells is often complicated by accompanying normal cells, representing the original tissue or infiltrating leukocytes. This holds particularly true for tissues with a great morphological and immunophenotypical variability, such as bone marrow. A method that identifies mitotic tumor cells by chromosomal aberrations and permits the subsequent immunophenotypical analysis was a first progress, demonstrated by Teerenhovi et al. However, the results are usually hampered by the low number of analyzable mitoses. We demonstrate here a method that simultaneously combines immunophenotyping and in situ hybridization with centromere-specific probes. Using our method, numerically aberrant tumor cells can be identified by interphase cytogenetics and subsequently analyzed immunophenotypically. Since all interphase cells can be analyzed, we are not limited by the number and banding quality of analyzable mitoses.
Lectin cytochemistry in the gastrointestinal tract with special reference to glycosylation in the Golgi apparatus of Brunner's gland cells.
Authors: Suzaki E , Kataoka K
Published: April 2011

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.
The use of silver-enhanced 1-nm gold probes for light and electron microscopic localization of intra- and extracellular antigens in skin.
Authors: Shimizu H , Ishida-Yamamoto A , Eady R A
Published: April 2011

We used colloidal gold (1-nm diameter) with silver enhancement, in conjunction with a low-temperature post-embedding immunolabeling technique, to localize several antigens in normal skin at both the light and the electron microscopic level within the same tissue blocks. Normal skin subjected to cyrofixation and cryosubstitution and embedded in Lowicryl K11M was used as a substrate. Semi-thin sections (1 micron) were incubated in primary antibody (against epidermal basement membrane zone associated antigens and two keratin sub-types), biotinylated secondary antibodies, and then in 1-nm gold-conjugated streptavidin. Finally, the 1-nm gold label was enhanced using silver staining. Labeling of both basement membrane and keratin antigens was well demonstrated, and the area in the semi-thin sections showing the best structural preservation and the greatest intensity of immunolabeling was used to identify the part of the block to be used for ultra-thin sectioning. Ultra-thin sections were treated using a similar procedure to that employed for semi-thin sections. The labeling with silver-enhanced 1-nm gold probes was intense and readily visible by electron microscopy, even at low magnification. We have found this technique to have a high degree of specificity and sensitivity for labeling both intra- and extracellular antigens in skin, with the added advantage of providing the means for studies at both light microscopic and electron microscopic level.
Identification of type I collagen-producing cells in human gastrointestinal carcinomas by non-radioactive in situ hybridization and immunoelectron microscopy.
Authors: Ohtani H , Kuroiwa A , Obinata M , Ooshima A , Nagura H
Published: April 2011

Invasive growth of cancer cells induces desmoplastic reaction as one of the host reactions. It has been a matter of controversy whether stromal collagen is produced by cancer cells or stromal fibroblasts. In the present study, we investigated the cellular origin of Type I collagen in human gastrointestinal carcinomas by in situ hybridization and immunoelectron microscopy. In situ hybridization technique with digoxigenin-labeled RNA probes revealed that spindle-shaped fibroblasts in the stromal area were abundantly positive for transcripts of pro alpha 1(I) collagen in intestinal-type adenocarcinoma. Gland-forming carcinoma cells were negative. In diffuse-type carcinoma of the stomach, spindle-shaped or stellate fibroblasts were positive, whereas dissociated, oval carcinoma cells were negative. A precise determination of cell type was done by immunoelectron microscopy. Intracellular immunoreactivity for Type I collagen was observed in rough endoplasmic reticulum of fibroblasts (including myofibroblasts) in the stromal area. No definite reactivity was obtained in cancer cells by either in situ hybridization or immunoelectron microscopy. Our results indicated that stromal Type I collagen is produced by stromal fibroblasts, which are activated by cancer invasion.
Electron microscopic localization of the multicatalytic proteinase complex in rat liver and in cultured cells.
Authors: Rivett A J , Palmer A , Knecht E
Published: April 2011

The multicatalytic proteinase (MCP) prosome or proteasome is a large multifunctional complex which is believed to play a major role in non-lysosomal pathways of intracellular protein degradation and has recently been implicated in antigen processing. In this study, affinity-purified antibodies against rat liver MCP were used to investigate the localization of the proteinase both in rat liver and in growing human L-132 cells in culture, using electron microscopic immunogold techniques. Quantitation of the MCP in different subcellular localizations by morphometric analysis of electron micrographs showed the proportion in the nucleus to be 17% for hepatocytes and 51% for L-132 cells, demonstrating differences in the distribution of MCP in different cell types. In hepatocytes, 14% of the total MCP was found associated with the endoplasmic reticulum. The remainder was localized in the cytoplasmic matrix. Immunofluorescence studies with L-132 cells also showed a reaction in nuclei and cytoplasm. The localization of MCP is consistent with its proposed multiple functions in protein turnover, in the production of peptides for antigen presentation, and in RNA processing.
Isolation of monoclonal antibodies recognizing rat bone-associated molecules in vitro and in vivo.
Authors: Turksen K , Bhargava U , Moe H K , Aubin J E
Published: April 2011

Knowledge of the number and kinds of differentiation steps that characterize cells of the osteoblast lineage is inadequate. To further analyze osteoblast differentiation, we generated a series of monoclonal antibodies (MAb) to osteogenic cells. Spleen cells from mice immunized with whole-cell populations enriched for expression of osteoblast-associated properties or bone formation in vitro were fused with the SP2/0 myeloma cell line. Supernatants from growing hybridomas were screened by indirect immunofluorescence on frozen sections of a portion of 21-day fetal rat heads that included the calvaria bone, periosteum, muscle, fibrous connective tissue, and skin. Six MAb were selected with bone-associated staining and limited ability to label other tissues. Either cell surface or cytoplasmic molecules were recognized by five of the MAb; one recognized a molecule detectable both in the cytoplasm, on the cell surface, and in the extracellular matrix. Of the antibodies selected, one identified both preosteoblasts and osteoblasts and has been found to be against alkaline phosphatase. The others recognized the mature osteoblasts, osteocytes, and chondrocytic cells. The pattern and distribution of the labeling in vivo extended to primary cells and cell lines in vivo. These results support earlier observations on molecules differentially expressed by cells at different stages of the osteoblast lineage and extend the available cell surface and cytoplasmic epitopes identifiable as marker molecules.