bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibodies specific to quail embryo tissues: their epitopes in the developing quail embryo and their application to identification of quail cells in quail-chick chimeras.
Authors: Aoyama H , Asamoto K , Nojyo Y , Kinutani M
Published: April 2011

Quail-chick chimeras have been used extensively in the field of developmental biology. To detect quail cells more easily and to detect cellular processes of quail cells in quail-chick chimeras, we generated four monoclonal antibodies (MAb) specific to some quail tissues. MAb QCR1 recognizes blood vessels, blood cells, and cartilage cells, MAb QB1 recognizes quail blood vessels and blood cells, and MAb QB2 recognizes quail blood vessels, blood cells, and mesenchymal tissues. These antibodies bound to those tissues in 3-9-day quail embryos and did not bind to any tissues of 3-9-day chick embryos. MAb QSC1 is specific to the ventral half of spinal cord and thymus in 9-day quail embryo. No tissue in 9-day chick embryo reacted with this MAb. This antibody binds transiently to a small number of brain vesicle cells in developing chick embryo as well as in quail embryo. A preliminary application of two of these MAb, QCR1 and QSC1, on quail-chick chimeras of neural tube and somites is reported here.
Immunohistochemical and immunoelectron microscopic analyses of alpha-amylase isozymes in human intrahepatic biliary epithelium and hepatocytes.
Authors: Terada T , Kono N , Nakanuma Y
Published: April 2011

The expression and localization of the pancreatic and salivary isozymes of alpha-amylase in the intrahepatic biliary epithelium and hepatocytes were examined by the immunohistochemical method with polyclonal and monoclonal antibodies in 45 normal autopsied human livers. Immunoelectron microscopic studies with the protein A-gold method were performed with the monoclonal antibodies (MAb) on seven of the livers. The intrahepatic biliary system was divided into large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands. Immunohistochemically, pancreatic isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands in almost all livers. Interlobular ducts expressed pancreatic isozyme in only four (9%) livers. Bile ductules and hepatocytes were negative for pancreatic isozyme in all cases. Expression of salivary isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands in almost all livers, although the expression in interlobular ducts and bile ductules was weak. Hepatocytes were weakly positive for salivary isozyme. Immunoelectron microscopy revealed that both pancreatic and salivary isozymes were located in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands, and that hepatocytes had no pancreatic isozyme but contained salivary isozyme. These data suggest that pancreatic and salivary isozymes of alpha-amylase are produced by the intrahepatic biliary epithelium and secreted into intrahepatic biliary lumens, and that they may play an important role in the physiology of the intrahepatic biliary tree and hepatic bile. It is also suggested that hepatocytes produce a small amount of salivary alpha-amylase that may be secreted into the biliary tree.
Demonstration of dopamine-immunoreactive cells in the proximal convoluted tubule of gerbil (Meriones unguiculatus) kidney.
Authors: Dawirs R R , Teuchert-Noodt G
Published: April 2011

We investigated dopamine immunoreactivity in the kidney of gerbils (Meriones unguiculatus). For that purpose a sensitive and selective antibody against glutaraldehyde-conjugated dopamine was applied. Dopamine-immunoreactive cells were found in the epithelium of the proximal convoluted tubule, where these cells revealed a typical segment-like distribution pattern. Dopamine-immunoreactive precipitates were small and concentrated at the apical pole of the labeled cells. This study has directly identified dopamine as a constituent of certain cells of the proximal convoluted tubule in gerbils. The functional significance of dopamine in these cells is discussed in relation to the present view of renal dopaminergic actions.
Complete penetration of antibodies into vibratome sections after glutaraldehyde fixation and ethanol treatment: light and electron microscopy for neuropeptides.
Authors: Llewellyn-Smith I J , Minson J B
Published: April 2011

To develop a method for quantitative electron microscopic immunocytochemistry on neural tissue of CNS, we tested the extent to which ethanol treatment would improve the penetration of immunoreagents through vibratome sections fixed in high concentrations of glutaraldehyde without compromising ultrastructure. Transverse or sagittal vibratome sections (60-80 microns) of spinal cord perfused with 1% formaldehyde plus 1% or 2.5% glutaraldehyde were washed in 50% ethanol for 0-70 min and stained to reveal immunoreactivity for neuropeptide Y (NPY). Semi-thin (1 micron) or ultra-thin sections were used to assess the depth to which NPY nerve fibers in the dorsal horn were stained. Without ethanol washing, immunoreactive nerve fibers were visualized only in the surface 5-10 microns of transverse or sagittal vibratome sections. In transverse vibratome sections, NPY nerve fibers, which ran perpendicular to the cut surfaces of the sections, were entirely stained after a 30-min wash in 50% ethanol. The numbers of NPY-immunoreactive varicosities and synapses were comparable at the surfaces and in the centers of the vibratome sections. In sagittal sections, where NPY nerve fibers ran parallel to the cut surfaces, fibers in the centers of vibratome sections could not be labeled even after 70 min in 50% ethanol. Substance P- and enkephalin (Enk)-immunoreactive nerve fibers could also be completely stained in transverse sections of spinal cord or medulla oblongata after 30-min exposure to ethanol. Ethanol washing had no significant deleterious effects on ultrastructure, although the amount of cytoplasmic matrix in neurons decreased with increasing exposure. These results indicate that washing with 50% ethanol for at least 30 min allows immunoreagents to penetrate completely through nerve fibers fixed with high concentrations of glutaraldehyde, as long as the fibers have cut ends at both surfaces of a vibratome section. This technique makes possible quantitative electron microscopic immunocytochemical studies and is proving a useful tool for defining synaptic connections in the CNS.
Nature and distribution of mineral-binding, keratan sulfate-containing glycoconjugates in rat and rabbit bone.
Authors: Maeno M , Taguchi M , Kosuge K , Otsuka K , Takagi M
Published: April 2011

The presence of keratan sulfate (KS) and KS proteoglycans in bone has been demonstrated in birds and rabbits but comparison with other animal species has not been investigated. The nature and distribution of mineral-binding, KS-containing glycoconjugates in rat and rabbit bone were investigated with a monoclonal antibody (MAb 5D4) specific for KS. Mineral-binding proteins were extracted from the mineralized bone with 0.4 M EDTA without guanidine-HCl (E-extract). On Western blot analysis of SDS-polyacrylamide gel electrophoresis, rat E-extract gave a weak 5D4-reactive band, M(r) 66,000-68,000, whereas rabbit E-extract produced two major reactive populations of small and large molecular size; one population consisted of two closely spaced bands at M(r) 61,000-63,000 and 66,000-68,000, and the other population consisted of one band at approximately M(r) 200,000. The identity of KS chains was further established by the sensitivity of these bands to keratanase II (Bacillus sp. Ks 36) and endo-beta-galactosidase. Immunocytochemistry with MAb 5D4 showed that, in rat bone, staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas the remainder of the mineralized matrix lacked staining. In contrast, in rabbit bone the staining was distributed over the entire portion of the mineralized matrix with focal accumulation of staining in the wall of the lacunocanalicular system. These results indicate that rat bone contains a mineral-binding, KS-containing glycoconjugate with preferential localization in the wall of the lacunocanalicular system, whereas rabbit bone contains at least two or possibly three types of KS-containing glycoconjugates distributed over the entire portion of the mineralized matrix.
Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.
Authors: Santisteban M S , Montmasson M P , Giroud F , Ronot X , Brugal G
Published: April 2011

This study is intended to be the first step of an in situ exploration of the intranuclear DNA distribution by image cytometry (SAMBA) with several fluorochromes. The nuclear DNA content and the chromatin pattern, revealed by ten fluorochromes (HO, DAPI, MA, CMA3, OM, QM, AO, EB, PI, and 7-AMD), were analyzed on mouse hepatocytes fixed by the Boehm-Sprenger procedure optimal for preserving the chromatin pattern. The question was whether fluorochromes specific to DNA make it possible to accurately quantitate the total nuclear DNA content when the chromatin pattern is preserved. Only HO and MA were found to provide satisfactory quantitation of nuclear DNA content, as assumed by both a small CV and a 4c to 2c ratio equal to 2. PI, EB, 7-AMD, and OM provided higher CV values, although the 4c to 2 c ratio was still equal to 2. QM, AO, CMA3, and DAPI provided non-reproducible and non-stoichiometric nuclear DNA content measurements under the fixation conditions used. The intranuclear and the internuclear SD of the fluorescence intensities describing the fluorescence pattern of the 2c hepatocytes proved to vary according to both the basepair specificity and the binding mode of the fluorochromes. The results reported here argue in favor of an external binding of 7-AMD to DNA and an increased quantum yield of QM when bound to AT-rich DNA. For PI, EB, 7-AMD, and OM, the measured DNA content increased with the fluorescence distribution heterogeneity. This correlation was not observed with other fluorochromes and is suggested to result from decreased fluorochrome accessibility to DNA when the chromatin is condensed. This study demonstrates that under conditions that preserve chromatin organization, only HO for AT-rich DNA and MA for GC-rich DNA can be used, alone or in combination, to measure nuclear DNA content. With other fluorochromes, either the measured DNA content or the chromatin pattern is assessed in suboptimal conditions when fluorescent image cytometry is used.
A new acrylic resin formulation: a useful tool for histological, ultrastructural, and immunocytochemical investigations.
Authors: Scala C , Cenacchi G , Ferrari C , Pasquinelli G , Preda P , Manara G C
Published: April 2011

We describe a new formulation for a hydrophilic resin, mostly composed of glycol methacrylate and hydroxypropyl methacrylate and here referred to as bioacryl, that allows the performance of morphological and immunohistochemical investigations at both light and electron microscopic levels. Immunolocalizations performed on bioacryl-embedded tissues are characterized by high specificity with virtually absent background staining. Finally, the new resin yields satisfactory fine-structural preservation, resulting in ultrastructural images of better quality than those obtained with Lowicryl K4M.
Loss of laminin epitopes during glomerular basement membrane assembly in developing mouse kidneys.
Authors: Abrahamson D R , St John P L
Published: April 2011

Kidney glomerular basement membranes (GMBs) originate in development from fusion of a dual basement membrane between endothelial cells and primitive epithelial podocytes. After fusion, segments of newly synthesized matrix, derived primarily from podocytes, appear as subepithelial outpockets and are spliced into GBMs during glomerular capillary loop expansion. To investigate GBM assembly further, we examined newborn mouse kidneys with monoclonal rat anti-mouse laminin IgGs (MAb) conjugated to horseradish peroxidase (HRP). In adults, these MAb strongly label glomerular mesangial matrices but bind only weakly or not at all to mature GBMs. In contrast, anti-laminin MAb intensely bound newborn mouse GBMs undergoing initial assembly. After intraperitoneal injection of MAb-HRP into neonates, dense binding occurred across both subendothelial and subepithelial pre-fusion GMBs as well as forming mesangial matrices. Considerably less MAb binding was seen, however, in post-fusion GBMs from more mature glomeruli in the same section, although mesangial matrices remained positive. In addition, new subepithelial segments in areas of splicing were negative. These results conflict with those obtained previously with injections of polyclonal anti-laminin IgGs into newborns or adults, which result in complete labeling of all GBMs. Although epitope masking cannot be completely excluded, we believe that decreased MAb binding to developing GBM reflects actual epitope loss. This loss could occur by laminin isoform substitution, conformational change, and/or proteolytic processing during GBM assembly.
Immunocytochemical and biochemical demonstration of formaldhyde dehydrogenase (class III alcohol dehydrogenase) in the nucleus.
Authors: Iborra F J , Renau-Piqueras J , Portoles M , Boleda M D , Guerri C , Pares X
Published: April 2011

Alcohol dehydrogenase (ADH), the major enzyme catalyzing the biological oxidation of ethanol in mammals, includes four classes with very different capacities for ethanol oxidation. Class III ADH is present in all the tissues and is well conserved throughout evolution. This enzyme has a low activity with ethanol, is specific for the glutathione-dependent oxidation of formaldehyde, and is therefore a formaldehyde dehydrogenase (FALDH). Until now there have been few and conflicting studies concerning its intracellular distribution, which is important for the understanding of its role in cell function. In the present work we used biochemical and immunocytochemical methods to assess the distribution of FALDH in rat hepatocytes and astroglial cells. With the glutathione-dependent formaldehyde dehydrogenase assay, we found the highest activity in the cytosol of hepatocytes and brain cells (12 and 2.6 mU/mg protein, respectively), but nuclei also exhibited significant activity (1.16 and 2.1 mU/mg protein, respectively). The immunocytochemical results showed the presence of FALDH binding sites in both the cytoplasm and the nucleus of the different cell types studied. Whereas no specific gold particle labeling was seen associated with any cytoplasmic component, in the nucleus the particles were found mainly over condensed chromatin and interchromatin regions. Finally, the gold particle density over both the nucleus and cytoplasm was greater in differentiated than in proliferating astrocytes in primary culture. In contrast, class I ADH, primarily responsible for ethanol metabolism, was found only in the cytoplasm of hepatocytes. We propose that one of the functions of FALDH is to protect cell structures, including DNA, from the toxic effects of endogenous formaldehyde, which is an intermediate in many metabolic process.
Iron content correlates with peroxidase activity in cysteamine-induced astroglial organelles.
Authors: McLaren J , Brawer J R , Schipper H M
Published: April 2011

A subpopulation of astrocytes in periventricular brain regions and in cysteamine-treated neuroglial cultures contains cytoplasmic granules that exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic peroxidase activity. The autofluorescence and pseudoperoxidase activity are consistent with the presence of porphyrins and heme iron, respectively. In the present study, we employed diaminobenzidine cytochemistry, transmission electron microscopy, and energy-dispersive X-ray microanalysis (electron microprobe) in an attempt to correlate fine structure with the peroxidase activity and elemental composition of the cysteamine-induced inclusions in cultured astrocytes. In osmicated preparations, these membrane-bound inclusions varied greatly in size, were round or ovoid in shape, and exhibited an intensely electron-dense granular matrix. In non-osmicated preparations, many inclusions exhibited internal membranous partitions producing complex subcompartmentalization. Diaminobenzidine reaction product, indicative of endogenous peroxidase activity, was occasionally observed distributed diffusely throughout the granule matrix. More commonly, peroxidase activity was restricted to specific intraorganellar compartments. Elemental iron was detected in the inclusions by electron microprobe analysis. The presence and concentration of iron in these organelles correlated closely with the presence and intensity of diaminobenzidine staining, suggesting that redox-active iron mediates the pseudoperoxidase reactions in these cells. Cysteamine-induced derangements of porphyrin-heme biosynthesis may be responsible for the proliferation of iron-containing gliosomes in these astrocytes.
Quantitation of gold labeling and estimation of labeling efficiency with a stereological counting method.
Authors: Lucocq J
Published: April 2011

The amount of immunolabeling over a cell compartment of an average cell was estimated by use of an adaptation of the double disector method introduced by Gundersen. The first and last sections of a stack of ultra-thin sections formed a disector in which cell number could be estimated and related to a defined reference volume to give the cell density. Another stack section, selected at random, was immunolabeled and the number of gold particles associated with unit volume reference space (gold "density") estimated in quadrats placed systematically across the section. The ratio of gold density to cell density was used to estimate the number of gold particles lying over a chosen compartment of an average cell, N(gold)/N(cell). Such estimates required neither cell volume nor section thickness measurement and were reproducible. By combining the number of gold particles per cell with estimates of the number of protein antigens per cell, the number of gold particles associated with each antigen could be found (labeling efficiency).
Immunolocation analysis of glycosaminoglycans in the human growth plate.
Authors: Byers S , Caterson B , Hopwood J J , Foster B K
Published: April 2011

Monoclonal antibodies were used in this study to immunolocate glycosaminoglycans throughout the human growth plate. Chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate were observed in the extracellular matrix of all zones of the growth plate and persisted into the cartilage trabeculae of newly formed metaphyseal bone. Also present in the extracellular matrix was an oversulfated chondroitin/dermatan sulfate glycosaminoglycan which appeared to be specific to the proliferative and hypertrophic zones of the growth plate. As with the other extracellular matrix molecules, this epitope persisted into the cartilage trabeculae of the metaphyseal bone. Zonal differences between the extracellular and pericellular or lacunae matrix were also observed. The hypertrophic chondrocytes appeared to synthesize chondroitin sulfate chains containing a non-reducing terminal 6-sulfated disaccharide, which were located in areas immediately adjacent to the cells. This epitope was not found to any significant extent in the other zones. The pericellular region around hypertrophic chondrocytes also contained a keratan sulfate epitope which was also observed in the resting zone but not in the proliferative zone. These cell-associated glycosaminoglycans were not found in the cartilage trabeculae of metaphyseal bone, indicating their removal as the terminal hypertrophic chondrocytes and their lacunae are removed by invading blood vessels. These changes in matrix glycosaminoglycan content, both in the different zones and within zones, indicate constant subtle alterations in chondrocyte metabolic products as they proceed through their life cycle of proliferation, maturation, and hypertrophy.
Acute suppression of albumin synthesis in systemic inflammatory disease: an individually graded response of rat hepatocytes.
Authors: Ballmer P E , Ballmer-Hofer K , Repond F , Kohler H , Studer H
Published: April 2011

The effects of an inflammatory insult on albumin of the rat liver were investigated at the cellular level and were correlated with serum albumin concentration. After SC injection of turpentine, the livers were perfused and fixed in vivo; serial liver sections were stained using a streptavidin-ABC-immunoperoxidase technique with an antibody to rat albumin. Albumin and total protein were measured at intervals after turpentine injection in whole livers and in serum. Fibrinogen was determined in plasma only. Twenty-four hours after turpentine injection serum albumin had dropped by 25% and was at 50% of its initial value at Day 3. Serum fibrinogen increased 2.4-fold within 24 hr and decreased thereafter. Liver homogenates showed no significant changes in albumin concentration. Immunohistochemically, all hepatocytes stained positive for albumin in normal animals. During inflammation, the immunostainable albumin content vanished entirely in a majority of all hepatocytes while remaining unchanged in other cells, thus producing a strikingly patchy staining pattern. No signs of resumption of albumin accumulation in depleted hepatocytes were seen after 8 days, despite a clear trend towards normalization of serum albumin concentration. These results suggest that individual hepatocytes differ widely in their response to agents that suppress albumin synthesis in an acute-phase reaction.
Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction.
Authors: Chiu K P , Cohen S H , Morris D W , Jordan G W
Published: April 2011

We developed a new method to amplify cell DNA in situ using the polymerase chain reaction (PCR). Proviral sequences of mouse mammary tumor virus (MMTV) contained in cultured cells and tissue sections were amplified intracellularly using a thermal cycler. Two techniques were employed to maintain the localization of the amplified DNA. First, complementary tails at the 5' ends of the oligonucleotide primers resulted in the synthesis of high molecular weight concatamers containing the target sequences. Second, the PCR was carried out in a thin film of agarose solidified over the tissue sections. The specifically amplified and localized DNA was then detected by in situ hybridization (ISH). Our results demonstrate that (a) DNA in tissue sections can serve as the target for the polymerase chain reaction in situ, (b) cell morphology is maintained, and (c) a target of 167 BP can be specifically detected in individual cells. This technique should be generally applicable to amplifying cellular DNA targets in tissue sections for detection in situ.
Simultaneous assessment of TGFB and cell cycle kinetics using IUdR/BrdU infusions in human neoplasms from plastic-embedded tissue.
Authors: Miller M A , Bokhari S A , Qadir K , Raza A
Published: April 2011

We describe an immunohistochemical technique that makes use of two monoclonal antibodies (MAb), one to detect the transforming growth factor B (TGFB) and another that reacts with iodo- and bromodeoxyuridine. The purpose of this technique is to determine the relationship between TGFB expression and the S-phase cells in human tumors. Since both can be distinctly identified in situ from tissue embedded in plastic, in assessment of the geographic orientation of S-phase cells in relation to such factors as TGFB, contiguity to blood vessels, nerve fibers, and macrophages can also be achieved.
A novel peptide-producing cell in Xenopus: multinucleated gastric mucosal cell strikingly similar to the granular gland of the skin.
Authors: Moore K S , Bevins C L , Tomassini N , Huttner K M , Sadler K , Moreira J E , Reynolds J , Zasloff M
Published: April 2011

We have characterized a novel peptide-containing cell within the gastric mucosa of Xenopus laevis. The cell is a spherical, multinucleated syncytial structure containing a cytoplasmic space filled with dense rice-shaped granules, and is strikingly similar in morphology to the well-studied granular gland of the amphibian skin. Immunohistochemical and immunogold methods were used to demonstrate that several peptides previously isolated from the granular glands of the skin, including the antimicrobial peptides magainin and PGLa (a peptide with amino-terminal glycine and carboxy-terminal leucinamide), are also stored in granules present in these enteric cells. These data demonstrate that this enteric peptide-producing cell is strikingly similar both morphologically and biochemically to the granular gland, previously considered a highly specialized structure of the amphibian integument. This novel gastric mucosal cell, which we have designated a "granular multinucleated cell," is distinct in its morphology and its diversity of stored peptide products from other well-characterized peptide-containing cells in the vertebrate gastrointestinal tract.
Localization of nonspecific lipid transfer protein (nsLTP = sterol carrier protein 2) and acyl-CoA oxidase in peroxisomes of pigment epithelial cells of rat retina.
Authors: Deguchi J , Yamamoto A , Fujiki Y , Uyama M , Tsukahara I , Tashiro Y
Published: April 2011

We investigated the localization of nonspecific lipid transfer protein (nsLTP) in rat retina, especially in the pigment epithelial (RPE) cells, by the avidin-biotin-peroxidase complex method on cryosections for light microscopy and by the cryoimmunogold method for electron microscopy. Light microscopic observation revealed that the RPE, inner segment layer, nerve fiber layer, and Müller cells contain nsLTP. In the RPE cells gold particles were exclusively concentrated in the small peroxisomes (microperoxisomes; 0.1-0.3 micron in diameter), which were identified by double staining using anti-nsLTP and anti-catalase antibodies. In the peroxisomes gold particles were distributed homogeneously in the matrices and no preferential binding to the limiting membrane was observed. Acyl-CoA oxidase was also localized in the matrices of the peroxisomes. We suggest that the peroxisomes in RPE cells play important roles in the metabolism of lipids of the outer segment disk membranes, especially in the beta-oxidation of polyunsaturated long-chain and very long-chain fatty acids, such as docosahexaenoic acid which is composed of approximately one third of fatty acids in the disk membranes.
A one-step cobalt-ferrocyanide method for histochemical demonstration of acetylcholinesterase activity in central nervous system tissue.
Authors: Schatz C R , Geula C , Morecraft R J , Mesulam M M
Published: April 2011

We introduce a one-step histochemical method with cobalt as the precipitating agent for ferrocyanide for the light microscopic demonstration of acetylcholinesterase activity. This method was used to demonstrate acetylcholinesterase in normal cortical fibers and neurons, as well as pathological elements such as plaques and tangles. This procedure can also be easily combined with immunohistochemical methods that use diaminobenzidine as a chromogen.
Retrograde tracing of zinc-containing neurons by selenide ions: a survey of seven selenium compounds.
Authors: Christensen M K , Frederickson C J , Danscher G
Published: April 2011

The autometallographic retrograde tracing of zinc-containing neurons by intracerebral injection of sodium selenite (Na2SeO3), introduced by Danscher in 1982, has recently been described in more detail. Intracerebral injections of both sodium selenide (Na2Se) and sodium selenite (Na2SeO3) have been successfully used; however, sodium selenite had a rather toxic effect on the injected tissue. In the present study, we tested seven different selenium compounds to find the most suitable compound for retrograde tracing of zinc-positive pathways. Among the tested compounds, sodium selenide (Na2Se) caused insignificant necrosis within the injection site and was easily transported retrogradely when handled anaerobically. Sodium selenide is therefore recommended as the compound of choice.
A fibroblast-associated antigen: characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells.
Authors: Rønnov-Jessen L , Celis J E , Van Deurs B , Petersen O W
Published: April 2011

Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts from vascular smooth muscle cells. The antigen was detected on the cell surface and in cathepsin D-positive and acridine orange-accumulating vesicular compartments of fibroblasts. Ultrastructurally, the antigen was revealed in coated pits and in endosomal and lysosomal structures. 1B10 recognized three major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10 immunoreactivity was specific to fibroblasts and smooth muscle differentiated fibroblasts within the context of vascular smooth muscle cells.
Carbohydrate patterns of the pure cholinergic synapse of Torpedo electric organ: a cytochemical and immunocytochemical electron microscopic approach.
Authors: Egea G , Marsal J
Published: April 2011

Using post-embedding gold staining techniques, we investigated the ultrastructural distribution of terminal sugars and carbohydrate chains located at the pure cholinergic electric organ tissue of Torpedo marmorata. Neither alpha-N-acetylgalactosamine (GalNAc)-specific lectins (DBA, SBA, HPA) nor monoclonal antibodies (MAb) recognizing Tn (Gal-NAc alpha-O-Ser/Thr; MAb Cu-1) and sialyl-Tn epitopes (NeuAc alpha 2,6GalNAc alpha-O-Ser/Thr; MAb B72.3 and OSM-10) were capable of labeling any of the synaptic structures. The absence of gold labeling was likewise noted with UEA-I (L-fucose) and with PNA (T-antigen, Gal beta 1,3GalNAc alpha). After neuraminidase pre-treatment of ultra-thin sections, PNA labeling was rendered evident, indicating the presence of neuraminic acid-masked T-antigen. Certain synaptic vesicles were labeled for neuraminic acid (LFA) and for N-acetyllactosamine (DSA), whereas others were not labeled at all. Gold labeling with LFA, RCA-I (beta-galactose), and DSA in the membrane infoldings of the dorsal face of the electrocyte was visualized. As noted above, the PNA reaction was revealed only after pre-treatment with neuraminidase. Dorsal (non-synaptic) basal lamina were reactive with DSA, whereas the synaptic portion was likewise labeled with LFA and RCA-I. Finally, RCA-I labeling was noted in the Schwann cell nucleus. Comparisons between these results and those described at the neuromuscular junction were made.
Widespread occurrence of chromogranins/secretogranins in the matrix of secretory granules of endocrinologically silent pituitary adenomas.
Authors: Rosa P , Bassetti M , Weiss U , Huttner W B
Published: April 2011

To investigate the constituents of the matrix of endocrine secretory granules, we analyzed endocrinoilogically silent ("non-functioning") human pituitary adenomas for the occurrence of the chromogranins/secretogranins (granins), a protein family normally stored together with many different hormones. When five non-functioning pituitary adenomas were analyzed by immunoblotting using polyclonal and monoclonal antibodies specific for individual members of the granin family, chromogranin A was detected in four cases and chromogranin B and secretogranin II were detected in all cases. The cellular distribution of the granins and of various hormones known to be expressed in the anterior pituitary was studied by immunocytochemistry in fixed, frozen tissue sections from five additional adenomas. Of the eight hormones investigated, only thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone were detected, occurring in only two of the five adenomas. In contrast, granins were found in all five tumors. Chromogranin B and secretogranin II were detected in each of the adenomas in virtually every cell studied, whereas chromogranin A exhibited such a widespread cell distribution in only three adenomas, being focally present in one and absent from the other tumor. The subcellular localization of the granins and the three glycoprotein hormones was investigated by double immunoelectron microscopy. Chromogranin A and chromogranin B were mainly co-localized in secretory granules, whereas secretogranin II was either co-localized with the other two granins or segregated to different secretory granules. When present, glycoprotein hormones were immunodetected in both the secretory granules containing all three granins and those containing mainly secretogranin II. Our data indicate that in non-functioning pituitary adenomas chromogranin A is differentially expressed from chromogranin B and secretogranin II. Moreover, the granins appear to be the most widespread constituents of endocrine secretory granules known, forming the dense-core matrix irrespective of the presence or absence of hormones.
Double labeling with non-isotopic in situ hybridization and BrdU immunohistochemistry: calmodulin (CaM) mRNA expression in post-mitotic neurons of the olfactory system.
Authors: Biffo S , Verdun di Cantogno L , Fasolo A
Published: April 2011

We describe a novel histochemical procedure for simultaneous detection of mRNA expression by in situ hybridization (ISH) and DNA synthesis on cells that are pulse-labeled with bromodeoxyuridine (BrdU) by immunohistochemistry (ICC). Pregnant rats were injected with BrdU at embryonic Day 20 and the olfactory bulbs of their pups were collected daily. The expression of calmodulin (CaM) mRNA was analyzed by ISH with an anti-sense digoxigenin-labeled riboprobe and BrdU incorporation by indirect ICC. Starting 5 days after BrdU injection, a few tufted and granular neurons of the olfactory bulb were observed to be double labeled for CaM mRNA and BrdU. To study the olfactory neuroepithelium, adult animals were injected with BrdU, sacrificed after 30 days, and the nasal mucosa dissected and decalcified. The co-expression of CaM mRNA and BrdU incorporation was then analyzed in the olfactory neuroepithelium: BrdU-positive primary olfactory neurons were also CaM mRNA positive. The combination of ISH and ICC on the same section resulted in improved BrdU staining with respect to both increased intensity and reduced background levels. The procedure described here can be applied to a variety of problems in developmental biology and is of potential value for correlating the timing of specific mRNA expression with the birth date of a cell type of interest.
Combination of immunocytochemistry and in situ hybridization in the same semi-thin sections: detection of Met-enkephalin and pro-enkephalin mRNA in the hypothalamic magnocellular dorsal nucleus of the guinea pig.
Authors: Mitchell V , Beauvillain J C , Mazzuca M
Published: April 2011

We describe a procedure for combining pre-embedding peroxidase immunocytochemistry with pre-embedding autoradiographic in situ hybridization in the same vibratome sections of paraformaldehyde-fixed brain tissue. The simultaneous detection of Met-enkephalin (Met-enk)-immunoreactive product and pro-enkephalin (PE) mRNA in neurons of the magnocellular dorsal nucleus (MDN) in the guinea pig hypothalamus was carried out as a model for this procedure. Vibratome slices were processed for Met-enk immunodetection followed by the incubation with a 45-base synthetic oligonucleotide complementary to PE mRNA labeled with 35S. Tissues were embedded in araldite, cut into semi-thin sections, and processed for autoradiography. Many neurons double labeled for Met-enk and PE mRNA were viewed in the MDN. The histological quality and the spatial resolution of both signals were optimized, since precise intracellular localization of hybridization sites was possible. This method allows simultaneous study of peptide immunoreactivity and mRNA expression levels in neurons within the same semi-thin sections. It may be useful for a variety of quantitative analyses, and might also be extended to ultrastructural analysis.
Enhanced synthesis of gelatinase and stromelysin by myoepithelial cells during involution of the rat mammary gland.
Authors: Dickson S R , Warburton M J
Published: April 2011

During the involution of the mammary gland there is destruction of the basement membrane as the secretory alveolar structures degenerate. Immunofluorescence staining of sections of rat mammary gland with antibodies to 72 KD gelatinase (MMP-2) and stromelysin (MMP-3) revealed increased production of these two proteinases during involution. This increased expression was mostly restricted to myoepithelial cells. Increased expression during involution was also demonstrated by immunoblotting techniques. Gelatin zymography indicated that the predominant metalloproteinase present in involuting rat mammary glands was a 66 KD gelatinase.
Histochemical staining of protein-tyrosine phosphatase activity in primary human mammary carcinoma: relationship with established prognostic indicators.
Authors: Kidd K R , Kerns B J , Dodge R K , Wiener J R
Published: April 2011

Protein-tyrosine phosphatase (PTPase) activity in frozen human mammary primary carcinoma tissue sections has been quantitated using a modified histochemical assay. The improved method features the assay of PTPase activity in 12-microns sections of air-dried unfixed tissues, and the use of [2-(N-morpholino)-ethanesulfonic acid] (MES) buffer to prepare stable reaction solutions. Tissue samples from 53 primary human mammary carcinomas were assayed for PTPase activity, and immunohistochemically stained for c-erbB-2 protein-tyrosine kinase expression. Elevated levels of PTPase activity were found in 68% of the tumors compared with the level of activity found in normal human mammary tissues. PTPase activity was co-localized with pathology definitive for carcinoma. Excessive activity was demonstrated throughout the cell, with high activity evident in the cell cytoplasmic membrane and the nucleus. Coexistence of elevated expression of c-erbB-2 and increased PTPase activity was present in 53% of the tumors. In contrast, 15% displayed low c-erbB-2 expression and high PTPase activity, and 24% displayed high c-erbB-2 expression and low PTPase activity. No statistically significant association was found between increased PTPase activity and either c-erbB-2 overexpression or grade and stage of disease in primary human mammary tumors.
Immunohistological demonstration of pyruvate kinase isoenzyme type L in rat with monoclonal antibodies.
Authors: Domingo M , Einig C , Eigenbrodt E , Reinacher M
Published: April 2011

Four monoclonal antibodies (MAb) specific for the L-type isoenzyme of rat pyruvate kinase (L-PK) were produced and characterized. They detect at least two different epitopes of the isoenzyme, as shown in interference binding assay and Western blot analysis after peptide mapping. The MAb were used in immunohistology to demonstrate the L-PK isoenzyme in paraffin-embedded normal rat tissues. L-PK was found only in hepatocytes, kidney proximal tubules, islet cells of pancreas, and epithelial cells of the villi of small intestine. The content of L-PK in hepatocytes was often lower in the periportal areas as compared with the periveneous zone. In kidneys a clearcut difference in L-PK content and distribution existed between male and female rats. Male animals possessed more L-PK in the kidney cortex than females. The L-PK content in the inner cortical zone (straight proximal tubules) was higher than in the outer cortical zone (convoluted proximal tubules) in kidneys of males. In contrast, female rats displayed less L-PK in the inner than in the outer cortical zone of the kidneys. Only some of them exhibited the same amount of the isoenzyme in both parts of the kidney proximal tubules.
Use of a double-label method to detect rapid changes in the rate of cell proliferation.
Authors: Hyatt G A , Beebe D C
Published: April 2011

We developed a double-label method to directly measure the rate at which cells enter S-phase of the cell cycle. All cells in S-phase were first labeled with a short pulse of [3H]-thymidine. This was followed by a longer incubation in bromodeoxyuridine (BrdU), a thymidine analogue. Nuclei labeled with [3H]-thymidine were detected by autoradiography and those labeled with BrdU by immunocytochemistry. Cells labeled only with BrdU must have entered S-phase at some time after the end of the [3H]-thymidine pulse. Thus, the rate of entry of cells into S-phase could be determined. This method was shown to be more accurate and more sensitive than determining changes in the rate at which cells entered S-phase with a continuous labeling protocol. It was possible to detect changes in proliferative activity that occurred in less than 1 hr. We used this double-label technique to study changes in the cell cycle during the terminal differentiation of chicken embryo lens fiber cells. These studies revealed differences in the effects of several treatments known to stimulate fiber cell differentiation. They also demonstrated the presence in the embryonic eye of factors that stimulate and prevent lens cell proliferation and differentiation.
Lectin activity of the major surfactant protein (SP-A) may participate in, but is not required for, binding to rat type II cells.
Authors: Thorkelsson T , Ciraolo G M , Ross G F , Whitsett J A , Morris R E
Published: April 2011

Pulmonary surfactant is a complex mixture of lipids and proteins, of which surfactant protein A (SP-A) is the most abundant glycoprotein. The SP-A molecule has several distinct structural features that include a lectin-like domain, sharing structural features with other mammalian lectins. We have tested the hypothesis that lectin activity of the SP-A molecule is required for the binding to its receptor on the surface of alveolar Type II cells. By using colloidal gold immunocytochemistry in conjunction with electron microscopy, we evaluated the ability of mannosylated proteins to inhibit canine SP-A binding to rat Type II cells in vitro. After preincubation of SP-A with the mannosylated protein horse-radish peroxidase (HRP), SP-A was incubated with isolated filter-grown Type II cells. HRP did not alter the binding of SP-A to the Type II cell surface. Evidence that SP-A did bind to HRP was shown by coincident observation of gold-labeled SP-A and HRP precipitates. These results provide visual evidence that the lectin activity associated with SP-A is not required for its binding to receptor on rat alveolar Type II epithelial cells.
Lectin binding patterns in normal, metaplastic, and neoplastic gastric mucosa.
Authors: Narita T , Numao H
Published: April 2011

We investigated lectin binding patterns on tissue specimens of normal and metaplastic gastric surface mucosae, gastric adenomas, and intestinal and diffuse-type gastric carcinomas. Compared with normal gastric mucosa, metaplastic mucosa exhibited an increase of ConA binding and decreases of WGA, PNA, UEA-1, and DBA binding in the cytoplasm, and decreases of ConA, PNA, and UEA-1 binding at the luminal surface. Intestinal carcinomas were similar to metaplastic gastric surface mucosa in ConA, WGA, and UEA-1 binding in the cytoplasm, while diffuse-type carcinomas were similar to normal gastric mucosa in WGA and UEA-1 binding in the cytoplasm. Adenomas were similar to intestinal carcinomas in ConA and UEA-1 binding in the cytoplasm, but were different from intestinal carcinomas in Con A and UEA-1 binding at the luminal surface. For UEA-1, normal and metaplastic gastric surface mucosae did not show a significant difference between the blood type A, AB, B group and the O group. Intestinal and diffuse carcinomas and adenomas also did not show such a difference between the blood groups. For DBA, normal gastric surface mucosa showed a significant difference between the blood type B, O group and the A, AB group. Normal gastric mucosa of the blood type A, AB group was frequently positive for DBA binding in the cytoplasm and at the luminal surface. Metaplastic mucosa did not show a significant difference between the blood groups. Intestinal and diffuse-type carcinomas and adenomas also did not show a difference between the blood groups. DBA binding in the cytoplasm of intestinal carcinomas and adenomas was more frequently positive than that of normal and metaplastic mucosae, except for normal gastric mucosa of the blood type A, AB group. Compared with diffuse-type carcinomas, intestinal carcinomas were accompanied by a significant increase of ConA binding and decreases of WGA and PNA binding in the cytoplasm.
In vivo determination of cell cycle kinetics of non-Hodgkin's lymphomas using iododeoxyuridine and bromodeoxyuridine.
Authors: Yanik G , Yousuf N , Miller M A , Swerdlow S H , Lampkin B , Raza A
Published: April 2011

Using sequential infusions of two S-phase-specific drugs, iododeoxyuridine and bromodeoxyuridine, we have developed an in vivo method for determining the labeling index (LI), the S-phase duration (Ts), and total cell cycle times (Tc) of non-Hodgkin's lymphomas. In nine non-Hodgkin's lymphomas studied, the LI ranged from 1.5% in a follicular small cleaved-cell lymphoma to 29.6% in a diffuse large-cell lymphoma. The Ts ranged from 16 hr in a large-cell lymphoma (immunoblastic type) to 117 hr in a follicular small cleaved-cell lymphoma. The Tc varied from 69 hr in a large-cell lymphoma (immunoblastic type) to over 1000 hr in all low-grade lymphomas studied. Immunohistochemical methods using anti-BrdU antibodies were used to detect cell incorporation of the two S-phase-specific drugs. In this manner, cell cycle times could be calculated while the architecture of the tumor specimen was preserved. Difficulties in using this methodology, specifically in the calculation of the growth fraction and total cell cycle times, are pointed out. This in vivo method does, however, allow for Ts calculations independent of growth fraction considerations. Correlations of cell cycle data with various biological and clinical factors await further patient follow-up.
Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry.
Authors: Berryman M A , Porter W R , Rodewald R D , Hubbard A L
Published: April 2011

We have modified our previous method for immunogold staining of unosmicated, plastic-embedded tissue by addition of tannic acid as a post-fixative to increase membrane contrast. Overall cell ultrastructure and organelle membranes, in particular, appeared well preserved after this treatment. We evaluated quantitatively the effect of tannic acid on the antigenicity of several membrane proteins in rat liver and intestine. For all antigens tested, significant antigenicity was retained on both intracellular and plasma membranes. However, the level of antigenicity decreased with increased concentrations of tannic acid. This effect was most apparent on the apical and basolateral membranes of hepatocytes and on the apical membrane of enterocytes, surfaces that had been in direct contact with the tannic acid fixative. The results indicate that when low concentrations of tannic acid are employed, this method yields greatly enhanced membrane contrast while preserving sufficient antigenicity to facilitate the ultrastructural localization of many membrane and other antigens.