bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoperoxidase localization of GCDFP-15 with mouse monoclonal antibodies versus rabbit antiserum.
Authors: Mazoujian G , Parish T H , Haagensen D E
Published: April 2011

Five monoclonal antibodies (Mabs) raised against separate determinants on a breast gross cystic disease fluid protein of 15 KD (GCDFP-15) were compared to one another and to a rabbit antiserum (Rb) against GCDFP-15 by radioimmunoassay (RIA) and by immunoperoxidase localization in paraffin-embedded tissues. All five Mabs and the Rb were equivalent in recognition of GCDFP-15 in solution, as determined by RIA. However, two of the Mabs (A5, B15) showed only minimal binding to GCDFP-15 in paraffin-embedded tissues, whereas the other three Mabs (B1, B4, D6) were equivalent to the Rb in staining intensity. These latter three Mabs and the Rb were evaluated by the immunoperoxidase technique on a variety of benign and malignant neoplasms as well as normal tissues (150 specimens) for staining specificity. Immunoperoxidase staining by the three Mabs vs the Rb was equivalent in apocrine glands, metaplastic apocrine epithelium of breast, and breast carcinomas with apocrine features. No staining of the Mabs or Rb was seen in the other tissue specimens.
Cytochemical localization of blood group substances in human salivary glands using lectin-gold complexes.
Authors: Nakajima M , Ito N , Nishi K , Okamura Y , Hirota T
Published: April 2011

We investigated localization of blood group antigens and their related substances in human labial salivary and submandibular glands by application of a post-embedding cytochemical staining procedure using lectin- or glycoprotein-gold complexes. Surgical tissue was obtained from 10 patients. Blood group-specific lectins, such as Dolichos biflorus agglutinin or Helix pomatia agglutinin (group A-specific), Griffonia simplicifolia agglutinin-I B4 (group B-specific), and Ulex europaeus agglutinin I (group H-specific) could recognize A, B, and H antigens, respectively, only in mature secretory granules (mature SG), which were found preferentially in cells in the late phase of the maturation cycle. In immature secretory granules (immature SG), which were found in cells in the early or middle phase of the maturation cycle, no binding with these lectins was observed. The Golgi complexes and endoplasmic reticula also were not labeled with these lectins. In blood group O and B secretors, blood group antigens were uniformly distributed throughout all the mature SG examined. However, in blood group A secretors, the distribution was heterogeneous, i.e., in some granules only H antigen was demonstrated, whereas in others both A antigens and a small amount of H antigens were detected. Among the blood group-nonspecific lectins, wheat germ agglutinin (WGA) was found to bind more preferentially to immature SG than to mature SG. This was demonstrated irrespective of the blood group and secretor status of the tissue donor, except that in blood group A secretors WGA bound strongly to some mature SG which possessed A antigen. We discuss the significance of cellular and subcellular mosaic distribution of blood group antigens in connection with morphological differences of secretory granules and the maturation cycle of mucous cells.
Localization of calcitonin and calcitonin gene-related peptide mRNAs in rat parafollicular cells by hybridocytochemistry.
Authors: Zabel M , Schäfer H
Published: April 2011

By use of appropriate fragments of CT DNA or a CGRP DNA and SP6 polymerase system, we produced anti-sense RNA probes labeled with biotinylated 11-UTP. The labeling and specificity of the RNA probes were confirmed using dot-blot hybridization. By use of hybridocytochemistry, CT mRNA and CGRP mRNA were localized in all parafollicular cells in control and dihydrotachysterin-pre-treated rats. We concluded that all parafollicular cells simultaneously produce both CT mRNA and CGRP mRNA, either under control conditions or after stimulation by dihydrotachysterin-induced hypercalcemia.
Cellular distribution and amount of chromogranin A in bovine endocrine pancreas.
Authors: Ehrhart M , Jörns A , Grube D , Gratzl M
Published: April 2011

We determined the cellular distribution and the amount of chromogranin A in endocrine cells of bovine pancreas using a polyclonal antibody against bovine adrenomedullary chromogranin A. The relative amounts of chromogranin A in the different cells of the endocrine pancreas were determined by computer-assisted analyses of the optical densities of the immunoreactivities in the stained sections. More than 80% of the immunoreactive chromogranin A was located in the pancreatic B-cells. In immunoblots of acid tissue extracts, only one chromogranin A band (MW 74 KD) was observed. Quantification of the immunoblots revealed that 3 micrograms of chromogranin A and 918 micrograms of insulin were present per gram pancreas (wet weight), equivalent to a molar ratio of 460 mumol chromogranin A per mol insulin.
Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue.
Authors: Hayashi Y , Koike M , Matsutani M , Hoshino T
Published: April 2011

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.
Catecholamine innervation of enkephalinergic neurons in guinea pig hypothalamus: demonstration by an in vitro autoradiographic technique combined with a post-embedding immunogold method.
Authors: Mitchell V , Beauvillain J C , Poulain P , Mazzuca M
Published: April 2011

To study the relationship between the catecholamine (CA) nerve endings and the enkephalinergic cell bodies in the magnocellular dorsal nucleus (MDN) of guinea pig hypothalamus, double-labeling experiments were performed on the same tissue section at the electron microscopic level. An in vitro autoradiographic (ARG) method for [3H]-norepinephrine (NE) or [3H]-dopamine (DA) was combined with a post-embedding immunogold cytochemical technique for Met-enkephalin (Met-enk) in colchicine-treated animals. Hypothalamic slices (450 micrograms) were perfused with [3H]-NE or [3H]-DA at the fluid-gas interface, then fixed by immersion with glutaraldehyde and osmic acid. Semi-thin sections processed from the thickness of the slices showed adequate penetration of the tracers to all parts of the tissue. Frontal sections permitted visualization of some CA-uptake structures distributed around the cells. At the ultrastructural level, preservation appeared good on about 60% of the thickness of slices, and [3H]-CA structures were easily distinguished. Ultra-thin sections were successively incubated with Met-enk and colloidal gold-labeled antisera, followed by ARG processing. At the electron microscopic level, the good integrity of the tissue made possible visualization of [3H]-CA nerve terminals making synaptic contacts with enkephalinergic perikarya. These results provide morphological evidence for direct catecholaminergic control of enkephalinergic neurons of the MDN.
Cytochemical changes in hepatocytes of rats with endotoxemia or sepsis: localization of fibronectin, calcium, and enzymes.
Authors: Kang Y H , McKenna T , Watson L P , Williams R , Holt M
Published: April 2011

Bacterial lipopolysaccharide (LPS) is known to be implicated in the pathogenesis of endotoxemia and septic shock. The liver is the first vital organ to exhibit pathological alterations in shock. The present studies include immunoelectron microscopic localization of tissue fibronectin and cytochemical localization of calcium and enzymes in hepatocytes of animals with LPS-induced endotoxemia or cecal ligation-induced septic shock. The results showed increased staining of fibronectin in the basal (perisinusoidal) surfaces and in the cisternae of rough endoplasmic reticulum and the Golgi complex of hepatocytes in rats with endotoxemia or septic shock. Intracellular calcium content was significantly increased in the LPS-treated or septic rats. Calcium pyroantimonate precipitate was deposited predominantly on the outer surfaces of the RER of hepatocytes. In addition, diminution or depletion of glycogen, reduction of catalase-containing peroxisomes, increase of G-6-Pase activity, and depletion of cytochrome c oxidase in many mitochondria were also observed in hepatocytes of experimental animals. The overall results suggest that LPS stimulates: (a) hepatic synthesis and secretion of fibronectin; (b) uptake of calcium by hepatocytes; and (c) G-6-Pase activity. LPS treatment also leads to reduced numbers of peroxisomes and depletion of cytochrome c oxidase.
Cellular and subcellular immunolocalization of L-glutamate decarboxylase in rat pancreatic islets.
Authors: Garry D J , Appel N M , Garry M G , Sorenson R L
Published: April 2011

The cellular and subcellular distribution of L-glutamate decarboxylase (GAD), the biosynthetic enzyme for gamma-aminobutyric acid (GABA), was determined immunohistochemically in rat pancreatic islet using light and electron microscopic techniques. The cellular distribution of GAD was determined at the light microscopic level using an elution/re-staining protocol and a computerized digital image processing technique. At this level of resolution, immunofluorescent GAD was observed to be co-localized with immunofluorescent insulin in the islet B-cells and absent in both the A-cells, which contained glucagon, and the D-cells, which contained somatostatin. Subcellular localization of GAD was determined using an electron microscopic, colloidal gold post-embedding protocol and was compared to insulin immunoreactivity in serial sections of the same B-cell. In the same islet B-cell, GAD immunoreactivity appeared predominantly in the extragranular cytoplasm, whereas insulin immunoreactivity was associated with the secretory granules. Quantitative analysis of GAD immunoreactivity in the B-cell revealed 15.3 +/- 1.8 gold particles/micron2 in the cytoplasm, 1.7 +/- 0.2 gold particles/micron2 in the secretory granules, and 0.4 +/- 0.4 gold particles/micron2 in the mitochondria. The results of this study, localization of the biosynthetic enzyme for GABA to the B-cell cytoplasmic compartment and its absence in the secretory granules which contain insulin, are compatible with the hypothesis that GABA functions as an intracellular mediator of B-cell activity.
Histological detection of messenger RNAs with biotinylated synthetic oligonucleotide probes.
Authors: Guitteny A F , Fouque B , Mougin C , Teoule R , Bloch B
Published: April 2011

We achieved histological detection of the messenger RNAs coding for vasopressin, calcitonin, or calcitonin gene-related peptide by using biotinylated synthetic oligonucleotides, and defined the technical parameters enabling optimal detection of these mRNAs. Oligonucleotides labeled by fixation of one biotin at their 5' end or by addition of a biotin-11-dUTP tail at their 3' end can be used to detect mRNAs, although the latter are more sensitive. Streptavidin-alkaline phosphatase revealed with nitroblue tetrazolium-bromo-chloro-indolyl phosphate as substrate makes possible detection of the biotinylated oligonucleotides. Increasing formaldehyde concentration in the fixative decreases the signal intensity; 1% formaldehyde fixation provides the most intense signal. Several controls, including those with addition of unlabeled oligonucleotides to the hybridization buffer, confirm the specificity of mRNA detection. The sensitivity of the biotinylated probes is identical or lower as compared to the corresponding radiolabeled oligonucleotides. Histological and subcellular resolution is greatly enhanced with biotinylated probes. The rat vasopressin probes stain magnocellular neurons in the supraoptic and paraventricular nuclei and, under optimal conditions, parvocellular neurons in the suprachiasmatic nucleus. Vasopressin mRNA is present in the cytoplasm of the cell bodies and in the roots of certain processes. Calcitonin and calcitonin gene-related peptide mRNA are found co-localized in the cytoplasm of the same tumor cells in human medullary thyroid carcinoma.
Immunohistochemical localization of fibronectin and chondronectin in canine articular cartilage.
Authors: Burton-Wurster N , Horn V J , Lust G
Published: April 2011

We compared the distribution of fibronectin and chondronectin within the matrix of canine articular cartilage. Fibronectin was found throughout the matrix as well as pericellularly. In contrast, chondronectin was observed predominantly associated with the cell or pericellular matrix. Interactions of these molecules with matrix components in the pericellular matrix probably differs, however, since concentrations of hyaluronidase which prevented detection of pericellular fibronectin allowed detection of chondronectin. Chondronectin and fibronectin were detected in osteoarthritic cartilage as well as in disease-free cartilage. Penetration of biotinylated fibronectin into cartilage from the external medium occurred only in osteoarthritic cartilage and proceeded only from the articular surface. Disease-free cartilage appeared to maintain a barrier to fibronectin penetration from the articular surface which was sustained even after the proteoglycan content was markedly depleted by incubation of cartilage with catabolin or lipopolysaccharide. In cartilage that was proteoglycan-depleted, the only detectable penetration of external fibronectin was from the cut surface.
Localization of follicle regulatory protein in the porcine ovary.
Authors: Fujimori K , Rodgers K E , Nakamura R M , Katt E , Yanagihara D L , diZerega G S
Published: April 2011

The granulosa cell secretes a protein (follicle regulatory protein: FRP) that affects the responsiveness of other follicles to gonadotropin stimulation. This protein was purified, partially characterized, and rabbit antisera as well as monoclonal antibodies were prepared against FRP. Fixed sections of porcine ovaries were prepared on slides and then incubated with the monoclonal antibody or polyclonal antisera and then incubated with either biotinylated mouse IgM or rabbit IgG antisera, respectively. These sections were then incubated with avidin conjugated to horseradish peroxidase, followed by substrate. Staining with both the monoclonal antibody and the antisera was present in the cytoplasm of granulosa cells of small- or medium-sized antral follicles. Staining distribution was localized preferentially to cells near the basal lamina; the antral granulosa cells of viable follicles did not stain. Neither primordial follicles nor pre-antral follicles (less than 300 microns in diameter) showed any positive staining. Thecal cells were not stained in follicles less than 5 mm in diameter, whereas some large follicles (greater than 5 mm) contained staining in the theca. In the latter, specific granulosa staining was only weakly positive with the polyclonal antibody and negative with the monoclonal antibody. Atretic follicles contained significant staining of all epithelial cells adjacent to the basal lamina by both the monoclonal and polyclonal antibody preparations. Staining of the luteal ovary by the monoclonal antibody was limited to the large luteal cells. These findings suggest that FRP is produced by the granulosa cells of porcine follicles at the stage of maturation corresponding to 0.5 mm in diameter. As the viable follicle increases in size, production of FRP in the granulosa is reduced below the detectable level when the follicle exceeds 5 mm in diameter. The main source of FRP during the luteal phase is the large cell of the corpus luteum.
Immunoelectron microscopic demonstration of S-100b protein-like in centriole, cilia, and basal body.
Authors: Uchida T , Endo T
Published: April 2011

We report here the ultrastructural localization of S-100b protein-like immunoreactivity in the centriole, cilia, and basal body. Duodenum and trachea of guinea pigs and rats were fixed and immunostained by the protein A-gold method. All centrioles, cilia, and basal bodies observed showed clear S-100b protein-like immunoreactivity. Specific colloidal gold particles were located over the microtubules in these cell organelles. However, other microtubules scattered throughout the cytoplasm were devoid of immunoreactivity. Although the functional significance of S-100b protein-like immunoreactivity in the centriole, cilia, and basal bodies remains to be elucidated, the present results introduce new perspectives into the investigation of localization and function of S-100 proteins.
Identification of lymphocyte subpopulations with a polymethine dye.
Authors: Kass L
Published: April 2011

Using the polymethine dye p-ethoxyphenyl-p-aminostyryl-1,3,3-trimethyl-3H-indolium chloride as an aqueous stain applied to specimens of peripheral blood or buffy coat fixed in FAA fixative, differential coloration of leukocytes was achieved using darkfield illumination. Neutrophils stained dark maroon and contained green granules, eosinophils contained bright blue granules, basophils revealed yellow and pink granules, and monocytes stained green with green and yellow vacuoles. In studies of purified lymphocyte subpopulations obtained in a cell sorter, T-helper cells stained red, T-suppressor cells were yellow orange, B-cells appeared yellow and often contained yellow annular structures in the cytoplasm, and natural killer (NK) cells stained green and contained large green granules. As a rapid screening technique for identification of T-helper and T-suppressor cells and their ratios in health and disease, the new polymethine stain may complement the more complex monoclonal antibody techniques for identification of these cells.
Specificity of riboprobes for intracellular RNA in hybridization histochemistry.
Authors: Williamson D J
Published: April 2011

The specificity of the probe for intracellular RNA during hybridization histochemistry is usually confirmed by loss of the signal following ribonuclease pre-treatment of the target. Interpretation of such controls is complicated when single-stranded (asymmetric) RNA probes (riboprobes) are used for in situ hybridization, since the probes themselves may be degraded by residual ribonuclease. A protocol using the Ca2+-dependent enzyme, micrococcal nuclease, is presented to circumvent these difficulties.
Complementary distribution of carbamoylphosphate synthetase (ammonia) and glutamine synthetase in rat liver acinus is regulated at a pretranslational level.
Authors: Moorman A F , de Boer P A , Geerts W J , van den Zande L , Lamers W H , Charles R
Published: April 2011

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.
Improvement of immunofluorescence for diagnosis of AIDS using laser microscopy.
Authors: Wick G , Hengster P , Böck G , Dierich M P
Published: April 2011

One of the most commonly used methods for demonstration of HIV antibodies is indirect immunofluorescence employing HIV-infected, CD4-positive lymphoid cell lines as antigenic substrate. Immunofluorescence with conventional optic equipment is reported to be slightly less sensitive than enzyme-linked immunoassay (ELISA). We have developed an immunofluorescence microscope which is equipped with an argon laser that has the advantages of much brighter fluorescence than conventional techniques, the prevention of fluorescence bleaching, and the possibility of distinguishing specific from nonspecific staining by comparative analysis of the kinetics of the bleaching curves. This microscope has now been used for demonstration of HIV antibodies in indirect immunofluorescence tests on the H9 lymphoid cell line, which is highly efficient in expressing HIV after infection. Titers of ELISA and Western blot-verified HIV-positive patients and appropriate normal controls were compared using four types of microscopic equipment, including the laser immunofluorescence microscope. The latter afforded significantly higher titers than those obtained with conventional immunofluorescence microscopes, and also made possible the distinction between specific and nonspecific staining.
Immunocytochemical localization of myosin in rabbit liver cell.
Authors: Ueno T , Yasuura S , Watanabe S , Hirose M , Sekine T , Namihisa T
Published: April 2011

It has been established that the liver cell possesses its own myosin which resembles other non-muscle myosins in subunit composition and in its dependence of actin-activated Mg2+-ATPase activity on light chain phosphorylation (Ueno T, Sekine T: Biochem Int, 1987;15:1205). We have raised a specific antibody against rabbit liver cell myosin. Immunoblot analysis has shown that the purified antibody reacts only with the heavy chain of liver cell myosin. The antibody did not react with rabbit skeletal muscle myosin or with smooth muscle myosin extracted from rabbit intestinal wall. Cryostat liver sections analyzed by indirect immunofluorescence microscopy showed a characteristic polygonal staining pattern, indicating that myosin is concentrated close to the plasma membrane, particularly in the region of bile canaliculi. Myosin therefore appears to be localized in the area where actin filaments are also abundant.
Light microscopic immunocytochemical demonstration of thyroid-stimulating hormone (TSH) receptors on normal rat thyroid cells.
Authors: Schmid K W , Jasani B , Morgan J M , Williams E D
Published: April 2011

We present a method for immunohistochemical demonstration of surface-associated thyroid-stimulating hormone (TSH) receptors on direct imprints of rat thyroid follicular cells at the light microscopic level, by use of dinitrophenyl (DNP)-labeled bovine TSH as the primary probe in a DNP-hapten sandwich staining (DHSS) procedure. The light microscopically invisible diaminobenzidine (DAB) product of the enzyme reaction was amplified with a DAB enhancement technique (silver amplification), which yielded a reliable and distinct light microscopically visible cell surface staining. The consistently negative control results after inhibition with TSH receptor-specific reagents provide evidence in support of immunohistochemical localization of surface membrane-associated TSH receptors.
Ultrastructural localization of ouabain-sensitive, K-dependent p-nitrophenyl phosphatase activity in monkey eccrine sweat gland.
Authors: Saga K , Sato K
Published: April 2011

We studied the electron microscopic localization of ouabain-sensitive, potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity of the Na K-ATPase complex in Rhesus monkey eccrine sweat gland by use of the one-step lead citrate method of Mayahara et al. (Histochemistry 1980; 67:125). Reaction product was observed predominantly in the cytoplasmic side of the basolateral membranes of clear (secretory) cells, especially in the interdigitating membrane folds in the basal labryinth, and were completely abolished by 10 mM ouabain or by removal of K+. Little or no enzyme activity was noted in membrane processes in the intercellular canaliculi and in the secretory coil lumen. Basolateral membranes of the dark cells also showed moderate enzyme activity. The myoepithelial cell membrane was devoid of reaction product, except in a few membrane processes arising from the inner aspect of myoepithelial cells. In the coiled duct, K-pNPPase activity was present predominantly in the entire cell membrane of the peripheral ductal cells. The predominantly basolateral distribution of Na-K-ATPase in the eccrine sweat secretory cells is consistent with the concept that a Na-K-Cl co-transport model may be involved in the mechanism of eccrine sweat secretion.
Demonstration of antigens at both sides of plasma membranes in one coincident electron microscopic image: a double-immunogold replica study of virus-infected cells.
Authors: Rutter G , Bohn W , Hohenberg H , Mannweiler K
Published: April 2011

We present here a procedure for obtaining high-resolution topographical information about the spatial distribution of antigens at both sides of isolated plasma membranes. HeLa cells grown on coverslips and infected with measles virus served as a model system. Virus glycoproteins appearing at the cell surface were demonstrated by tagging them with rabbit anti-measles antibodies and protein A-gold probes. Cells were stabilized with tannic acid, covered with a cationized coverslip, and then split in potassium-containing buffer. Membranes adherent to the cationized coverslip were fixed in formaldehyde-glutaraldehyde and reacted with mouse monoclonal antibodies against various structural proteins of measles virus. Antibody binding sites at the cytoplasmic surface were visualized either by the antibody bridge method, using normal mouse Ig coupled to gold colloid of different sizes, or by the peroxidase-antiperoxidase procedure. After osmication and critical point-drying, the cytoplasmic surfaces were replicated by platinum-carbon evaporation and examined by TEM without prior cleaning from biological material. This new method permits concomitant localization of antigens present at the inner and outer leaflets of the plasma membrane, and provides high-resolution information about the three-dimensional organization of the cytoplasmic surface.
Monoclonal antibodies as probes for plasma membrane domains in the exocrine pancreas.
Authors: De Lisle R C , Logsdon C D , Hootman S R , Williams J A
Published: April 2011

Monoclonal antibodies (mAb) were generated as probes for the plasma membrane domains of pancreatic acinar cells. Primary monolayer cultures of mouse pancreatic acinar cells, which have an expanded apical surface relative to normal pancreas, were used to immunize rats. With conventional immunization and fusion protocols, 3% of the hybridomas were positive against the acinar lumen by indirect immunofluorescence of mouse pancreas cryosections. Culturing of spleen cells from an immunized rat on the apical surface of acinar cell monolayer cultures before fusion with the myeloma (an in vitro boost) doubled the percentage of hybridomas producing apical membrane-specific mAb. Monoclonal antibodies were characterized by immunofluorescence, ultrastructural immunoperoxidase cytochemistry, immunoprecipitation, and immunoblotting. One antibody, acinar-1 (IgG2a), labeled the apical membranes of pancreatic acinar cells, hepatocytes, salivary and lacrimal gland acinar cells, and the brush border of small intestine enterocytes. This mAb precipitated and blotted a protein of 94 KD. Acinar-2 (IgM) also labeled pancreatic acinar cell apical membranes but did not label other tissues and did not precipitate or blot. Acinar-3 labeled pancreatic acinar cell lateral membranes. Duct-1 (IgM) labeled pancreatic duct apical membrane and ducts in liver and salivary glands but did not precipitate or blot. These domain-specific mAb demonstrate that common antigenic determinants occur in the apical surfaces of several exocrine epithelia and may be important in secretion.
Correlation between immunochemical characteristics and immunohistochemical applicability of nine lines of monoclonal antibodies against rat brain glutaminase.
Authors: Kaneko T , Urade Y , Mizuno N
Published: April 2011

Nine lines of monoclonal IgM antibodies to rat brain glutaminase were produced from a mouse. The antibodies were named MAb-19, -21, -45, -48, -51, -55, -59, -97, and -120. Immunotitration tests revealed that six of the nine antibodies (MAb-19, -21, -45, -48, -97, and -120) absorbed dose-dependently more than 75% of glutaminase activity in rat brain. By immunoelectroblotting after gel electrophoresis of the homogenate, all antibodies showed a positive band at the same position as that of the purified enzyme. However, MAb-55, -59, and -97 crossreacted with other proteins. Despite the difference in their specificities, all of the antibodies except MAb-51 competed with one another for determinants on the enzyme. The competition test further indicated that the affinities of the antibodies were in the order MAb-120 greater than -21 greater than -48 greater than -19 not equal to -45 greater than or equal to -97 greater than or equal to -55 greater than or equal to 59. When applied to immunohistochemical staining of cerebral cortex of rat brain, the five specific antibodies (MAb-19, -21, -45, -48, and -120) produced essentially the same pattern of neuronal labeling; they labeled specifically pyramidal neurons, which are reportedly glutamatergic. The most intense labeling was obtained with MAb-120.
Developmental studies on expression of monoclonal antibody-defined cytokeratins by thymic epithelial cells from normal and autoimmune mice.
Authors: Savino W , Dardenne M
Published: April 2011

A major component of the thymic microenvironment is a network of thymic epithelial cells (TEC) which are able to express class II major histocompatibility complex products and to secrete thymic hormones. In the present investigation, we used a panel of anti-cytokeratin (CK) antibodies to establish distinct cytokeratin-defined TEC subsets. Four subpopulations were identified. One, in the cortex, is defined by anti-CK8 and anti-CK18 monoclonal antibodies (MAb). The other three subsets are medullary, two minor ones respectively reactive with anti-CK19 and KL1 monoclonal antibodies (the latter being specific for CK3 and 10), and a major one characterized by negative reaction with the above-mentioned MAb but strongly positive after labeling with a polyclonal (and polyspecific) anti-keratin immunoserum. Ontogenetic studies revealed that the CK8+/18+ TEC subset is the first to be detected in fetal life. Moreover, the numbers of CK3/10+ cells and CK19+ cells decrease in aging normal mice, a phenomenon that seems to occur early in autoimmune mice. We also observed that these two medullary TEC subsets are sensitive to high-dose in vivo treatment with hydrocortisone, which stimulates a dramatic increase in CK3/10+ cells and a certain decrease in CK19+ cells. Our results indicate that a number of mouse TEC subsets can be distinguished by cytokeratin expression. Such a strategy can be applied to analyze TEC sensitivity to drugs and might also be useful to further understanding of differential TEC function regarding intrathymic T-cell differentiation.
Immunohistochemical localization of Na+,K+-ATPase in rodent and human salivary and lacrimal glands.
Authors: Winston D C , Hennigar R A , Spicer S S , Garrett J R , Schulte B A
Published: April 2011

The enzyme Na+,K+-ATPase was localized immunohistochemically in major salivary glands of mouse, rat, and human and in exorbital lacrimal glands of the rodents. Immunoreactive Na+,K+-ATPase was abundant in the basolateral membranes of all epithelial cells lining striated and intra- and interlobular ducts of all glands. Reactivity of intercalated ducts varied among gland type and species. Cells lining granular ducts in rodent submandibular gland showed a heterogeneous staining pattern in rat but stained homogeneously in mouse. Secretory cells varied greatly in their content of immunoreactive Na+,K+-ATPase. As with all duct cells, staining was present only at the basolateral surface and was never observed at the luminal surface of reactive secretory cells. Mucous cells failed to show any reactivity in any gland examined. Serous cells showed a gradient of immunostaining intensity ranging from strongly positive in demilunes of human sublingual gland to negative in rat submandibular gland and lacrimal glands of rats and mice. The presence of basolaterally localized Na+,K+-ATPase in most serous cells but not in mucous cells suggests that the enzyme contributes to the ion and water content of copious, low-protein serous secretions. The intense immunostaining of cells in most if not all segments of the duct system supports the idea that the ducts are involved with modification of the primary saliva, and extends this concept to include all segments of the duct system.
Ultrastructural analysis of cytochrome oxidase in chick epiphyseal growth plate cartilage.
Authors: Yamamoto T , Gay C V
Published: April 2011

Histochemical detection of cytochrome oxidase activity in chicken growth plate revealed both positively and negatively stained mitochondria in chondrocytes of all zones, i.e., proliferative, pre-hypertrophic, hypertrophic, and calcifying zones. The proportion of positive to negative cells was lowest in the proliferative zone. As cytodifferentiation progressed, more positively stained cells were present. In positive cells all mitochondria were usually stained, and in negative cells all mitochondria were unstained. A few cells appeared to be in transition and contained both types of mitochondria. The results indicate that chondrocytes utilizing both aerobic and anaerobic metabolism are present in growth plate cartilage and that oxidative metabolism is favored in the more mature cells. The relationship of oxidative metabolism to calcification is discussed.
Detection of receptors for sulfated polysaccharides in human placenta by biotinylated probes.
Authors: Debbage P L , Lange W , Hellmann T , Gabius H J
Published: April 2011

Histochemical detection of binding sites for sulfated polysaccharides believed to be important mediators within recognitive interactions was carried out by application of biotinylated probes such as heparin, native and desulfated fucoidan, dermatan sulfate, and two types of carrageenans. The probes were derivatized by mild cyanogen bromide activation and subsequent aminoalkylation to allow incorporation of biotin, inserted with an epsilon-aminocaproic acid spacer to reduce charge-related and steric impediments. Specific labeling could be detected in different cell types of human placenta, dependent on the developmental stage. Sulfated polysaccharides bound predominantly to leucocytes in full-term placenta, whereas demonstration of specific binding sites in decidua, syncytiotrophoblasts, and cytotrophoblasts was restricted primarily to heparin and, less intensely, fucoidan, although not desulfated fucoidan. Heparin binding in the placenta after 8 weeks of gestation was reduced for epithelia that, at this stage of development, revealed carrageenan binding sites. Fucoidan binding was at this developmental stage measurable only for leucocytes. These results provide definite histochemical evidence for the presence and developmental regulation of expression of receptors for sulfated polysaccharides in different cell types of human placenta.
A reliable method combining horseradish peroxidase histochemistry with immuno-beta-galactosidase staining.
Authors: Sakanaka M , Magari S , Shibasaki T , Shinoda K , Kohno J
Published: April 2011

A sensitive combination of horseradish peroxidase (HRP) tracing and immunohistochemistry was used by Rye et al. [J Histochem Cytochem (1984) 32:1145] in a search for the origins of neurotransmitter- and neuromodulator-containing nerve fibers in brain. In this combination, peroxidase as a marker in immunohistochemistry was thought to yield a homogeneous brown immunoreaction product of diaminobenzidine, different from the black granular reaction product of retrogradely transported HRP, which is visualized by the tetramethylbenzidine (TMB) reaction and subsequent stabilization. A neuron that exhibits both kinds of reaction products in its cytoplasm in sections subjected to combination staining is referred to as a double-labeled cell. With a combined HRP and corticotropin-releasing factor (CRF) immunoperoxidase-antiperoxidase (PAP) method, the first set of experiments showed "false" double-labeled cells in the pyramidal cell layer of rat cerebral cortex, but only rarely in the subcortical areas, possibly because of the use of one enzyme system in two different histochemical procedures. This limitation of the double-staining technique prompted us to demonstrate an alternate combination of HRP tracing and immunohistochemistry in the second set of experiments by employing two previously described independent enzyme systems: HRP as a retrograde tracer and beta-galactosidase as a marker for immunohistochemical demonstration of CRF. A homogeneous blue reaction product indicated immuno-beta-galactosidase staining, and a granular black or brown reaction product labeled retrogradely transported HRP in double-labeled cells in subcortical regions. Neither double labeling nor "false" double labeling was seen in pyramidal cells of cerebral cortex. These findings suggest that application of two independent enzyme systems in a combined HRP and immunohistochemical method may be useful for investigating in origins of peptidergic fibers in brain when the combination of HRP histochemistry and the PAP method appears to be inappropriate.
Biotinylation of bacterial lipopolysaccharide and its applications to electron microscopy.
Authors: Odeyale C O , Kang Y H
Published: April 2011

We describe a procedure for lipopolysaccharide (LPS) biotinylation using N-biotinyl-L-lysine and application of the biotinylated LPS (Bi-LPS) to localization of LPS binding sites and subcellular distribution. Biotinylation of LPS was confirmed by enzyme-linked immunosorbent assay (ELISA), gel immunodiffusion, and immunodot techniques. The biological and toxicological activity of the Bi-LPS was tested by Limulus amoebocyte lysate (LAL) assays and histopathological examinations, respectively. Results showed that biotin was conjugated to LPS without disrupting the biological/toxicological activity of the molecule, which indicates that the biotin is directly linked to the polysaccharide portion of LPS. Localization of binding sites and subcellular distribution of Bi-LPS in human platelets and monocytes were studied by electron microscopy using an avidin-biotin-horseradish peroxidase (HRP) or streptavidin-gold method. Platelet surfaces were intensely stained by the reaction product of horseradish peroxidase (HPR) 5 min after incubation, and Bi-LPS was localized in small vesicles and vacuoles of platelets and in the phagocytic vacuoles of monocytes 60 min post incubation. Bi-LPS provides a reliable, stable, and sensitive tool for determination of LPS binding sites and subcellular distribution.
An immunohistochemical study performed with monoclonal and polyclonal antibodies to mouse epidermal growth factor.
Authors: Beerstecher H J , Huiskens-van der Meij C , Warnaar S O
Published: April 2011

We developed two monoclonal antibodies, E5 and F5, which react with mouse epidermal growth factor (mEGF) and urogastrone. These antibodies and a rabbit antiserum to mEGF were used for immunohistochemical analysis of staining reactions in rodent and human tissues. Our results do not confirm the published reports of EGF-like immunoreactivity in Brunner's glands, human submandibular glands, human kidney tissue, or rodent brain sections.
Alpha naphthyl acetate esterase activities in guinea pig Kurloff cells: a cytochemical and electrophoretic study.
Authors: Buat M L , Landemore G , Izard J
Published: April 2011

We established the presence of nonspecific esterases in the Kurloff cell (KC) by cytochemical methods at both light and electron microscope levels. Acid alpha-naphthyl acetate esterase (ANAE) activities were localized on the external face of the plasma membrane and on the external surface of the membrane surrounding the Kurloff body. Different cytosoluble KC extracts were obtained from purified splenic KC suspensions. About 18 isoenzymes were observed by isoelectric focusing, whereas after polyacrylamide gradient gel electrophoresis in native conditions almost all activity was observed on a few broad bands with very high apparent molecular weights, suggesting their oligomeric arrangement. After a first aqueous extraction step which released only a few isoenzymes, the remaining pellet was subjected to Triton X-100. This released almost all the isoenzymes observed after direct Triton X-100 extraction. These data suggest that almost all the KC esterases are membrane-bound enzymes, in agreement with the subcellular enzyme distribution. Different substrates were also used to characterize the different specificities of the KC isoesterases. Weak activity was detected with alpha-naphthyl butyrate by light cytochemistry, which essentially corresponded, on zymograms, to the membrane-bound esterase activity.
Phalloidin-gold complexes: a new tool for ultrastructural localization of F-actin.
Authors: Lachapelle M , Aldrich H C
Published: April 2011

We used a phalloidin-gold complex to study the distribution of F-actin in the myxamoebae and macroplasmodia of the slime mold Physarum polycephalum. After incubation of Lowicryl- or Quetol-embedded specimens with this complex, significantly different labeling intensities were found over the various cytoplasmic and nuclear regions of the cells. The nucleoplasm was the most heavily labeled cell compartment, followed in decreasing order of labeling intensity by the cytoplasm, the nucleolus, and the chromocenters. The labeling observed over the latter area did not appear significantly different from that of the background. Sections incubated in the phalloidin-gold complex to which an excess of F-actin was added showed no significant labeling over any of the above-mentioned cell regions. Other control experiments included incubation of the sections with a phalloidin solution followed by the phalloidin-gold complex, PEG-stabilized colloidal gold, and a bovine serum albumin-gold complex. There was no or very little labeling of the preparations.
Tartrate-resistant acid ATPase as a cytochemical marker for osteoclasts.
Authors: Andersson G N , Marks S C
Published: April 2011

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.
Characterization of a monoclonal antibody, OVB1, which binds to a unique determinant in human ovarian carcinomas and myeloid cells.
Authors: Kurrasch R H , Rutherford A V , Rick M E , Gallo M G , Lovelace E T , Pastan I , Willingham M C
Published: April 2011

A monoclonal antibody, OVB1, was generated against a human ovarian carcinoma cell line, OVCAR-3. The antigen reacting with this antibody was strongly expressed on the external surface of the plasma membrane of OVCAR-3 cells and cells of 4/4 other ovarian carcinoma lines. Variable density and homogeneity of expression was found on cells from 5/5 breast carcinoma lines. Various ovarian tumor specimens and normal human tissues were frozen, cryostat-sectioned, and examined for OVB1 reactivity using immunoperoxidase methods. A strong, uniform, homogeneous reaction on 10/10 ovarian carcinoma specimens and variable, non-homogeneous reactions on breast tumors were seen. Normal tissues reacting with the antibody include thyroid, pituitary pars intermedia, breast ductal epithelium, Auerbach's plexus and neuronal processes in the GI tract, colonic mucosal epithelium, and salivary gland ductal epithelium. Polymorphonuclear leukocytes, eosinophils, and approximately 13% of peripheral lymphocytes, as well as cells around germinal centers in lymph nodes and spleen, showed strong reactivity by immunofluorescence and/or immunoperoxidase. Expression of the OVB1 antigen in the myeloid cells of normal human bone marrow occurred from the promyelocyte stage through to more mature cells in a subpopulation of myeloblasts. Indirect immunofluorescence of live peripheral blood cells showed localization to the surface of PMNs, eosinophils, and certain lymphocytes. Double-immunofluorescence studies (with a direct fluorescein-anti-lactoferrin antibody conjugate) showed co-localization of OVB1 and OKM1 (anti-C3bi receptor) antibodies to specific granules of PMNs. Localization of OVB1 and OKM1 antibodies to granular structures in the PMN was confirmed by electron microscopy using the ferritin bridge technique. The antigen reacting with the OVB1 antibody was shown to be neuraminidase sensitive, but protease insensitive. The OVB1 monoclonal antibody may be useful in identification of ovarian tumors and subclassification of myeloid leukemias.
Formaldehyde-amine fixatives for immunocytochemistry of cultured Xenopus myocytes.
Authors: Luther P W , Bloch R J
Published: April 2011

Although formaldehyde is commonly used in immunocytochemical studies, this fixative can cause distortions in cell structure. We tested the possibility that adducts of formaldehyde and primary amines could be used as improved fixatives for immunolabeling studies of cultured cells. A variety of primary amines were reacted with formaldehyde and applied to cultured Xenopus muscle cells, after which the cultures were labeled for immunofluorescence. Amine-formaldehyde fixatives improved structural preservation of the myocytes as compared with formaldehyde alone. The extent of improvement depended on the amine tested; the best results were obtained using cyclohexylamine. Immunofluorescence localization of a variety of antigens was better in myocytes fixed with cyclohexylamine-formaldehyde than in cells fixed with formaldehyde alone. In addition, the fixative provided good ultrastructural preservation of cytoskeletal structures and permitted immunogold labeling for alpha-actinin by use of pre-embedding labeling techniques.
Detection of intracytoplasmic immunoglobulin by flow cytometry in B-cell malignancies.
Authors: Bardales R H , Al-Katib A M , Carrato A , Koziner B
Published: April 2011

The aim of this study was to compare the results of flow cytometric (FCM) determination of heavy and light chain cytoplasmic immunoglobulin (cIg) with those obtained by the peroxidase-antiperoxidase (PAP) method. Fifty-one patients, including five non-T-acute lymphoblastic leukemias, 16 B-chronic lymphocytic leukemias (CLL), 13 non-Hodgkin's lymphomas, seven hairy cell leukemias, four multiple myeloma/plasma cell leukemias, and six T-cell leukemia/lymphomas, as well as 12 normal controls, were studied. Saponin-permeabilized cell suspensions were indirectly stained with monoclonal antibodies and analyzed by flow cytometry. Acetone-fixed cytocentrifuge smears were stained for cIg by the PAP method. The results obtained indicate that: (a) detection of cIg by FCM is a feasible and useful technique to confirm the B-cell lineage of leukemias and lymphomas, particularly those characterized by low-density surface immunoglobulin, such as CLL; and (b) cIg detection by FCM and PAP staining are complementary methods to recognize with certainty the monoclonality of B-cell malignancies.