bluuebunny/crossref_metadata_2025 huggingface dataset:
A New Blocking Method for Application of Murine Monoclonal Antibody to Mouse Tissue Sections
Authors: Lu Qi L. , Partridge Terry A.
Published: April 2011

SUMMARY Antigen detection with primary antibody of the same species as the test tissue is complicated by high levels of background staining when indirect immunohistochemical detection methods are used. This severely limits the use of murine monoclonal antibodies on tissues of the mouse, the most widely used experimental model system; no method for blocking this is fully satisfactory. Here we show that background staining encountered in this system results largely from the binding of secondary antibodies via both Fc and Fab to endogenous immunoglobulins and other tissue components. A simple and efficient blocking strategy was established, employing papain-digested whole fragments of unlabeled secondary anti-mouse Igs enriched with Fc fragment of the same Igs. We have used this method to visualize dystrophin, an antigen expressed at low level, in revertant fibers of mdx mouse by both immunoperoxidase and immunofluorescence methods. In combination with the use of a biotin-streptavidin immunohistochemical detection protocol with biotinylated anti-mouse F(ab')2 as second layer, we eliminated the heavy background in this system and achieved strong signal amplification to demonstrate the specific antigen clearly. Double labeling with one mouse antibody and one antibody from another species was performed without signal interference. This principle can be adapted for wider applications, such as antibodies of other species on homologous tissues and perhaps where high background is found with heterologous antibodies.
The Distribution of the Matricellular Protein Thrombospondin 2 in Tissues of Embryonic and Adult Mice
Authors: Kyriakides Themis R. , Zhu Yu-Hong , Yang Zhantao , Bornstein Paul
Published: April 2011

Mice that lack the matricellular protein thrombospondin 2 (TSP2) develop a pleiotropic phenotype characterized by morphological changes in connective tissues, an increase in vascular density, and a propensity for bleeding. Furthermore, dermal cells derived from TSP2-null mice display adhesion defects, a finding that implicates TSP2 in cell-matrix interactions. To gain a better understanding of the participation of TSP2 in the development and maturation of the mouse, we examined its distribution in embryonic and adult tissues. Special attention was paid to the presence of TSP2 in collagen fibers, because collagen fibrils in the TSP2-null mouse appear to be irregular in size and contour by electron microscopy. Immunohistochemical analysis of Day 15 and Day 18 embryos revealed TSP2 in areas of chondrogenesis, osteogenesis, and vasculogenesis, and in dermal and other connective tissue-forming cells. Distinctly different patterns of deposition of TSP2 were observed in areas of developing cartilage and bone at Days 15 and 18 of embryonic development. A survey of adult tissues revealed TSP2 in dermal fibroblasts, articular chondrocytes, Purkinje cells in the cerebellum, Leidig cells in the testis, and in the adrenal cortex. Dermal fibroblasts were also shown to synthesize TSP2 in vitro. The distribution of TSP2 during development is in keeping with its participation in the formation of a variety of connective tissues. In adult tissues, TSP2 is located in the pericellular environment, where it can potentially influence the cell-matrix interactions associated with cell movement and tissue repair.
Changing Patterns of Renal Retinal Dehydrogenase Expression Parallel Nephron Development in the Rat
Authors: Bhat Pangala V. , Marcinkiewicz Mieczyslaw , Li Yuan , Mader Sylvie
Published: April 2011

We have recently characterized a cytosolic aldehyde dehydrogenase from rat kidney that functions as a retinal dehydrogenase (RALDH) and have cloned the corresponding gene. RALDH catalyzes the oxidation of retinal to retinoic acid, which regulates cell growth and differentiation by activating retinoic acid receptors. In situ hybridization demonstrates that RALDH mRNA expression is prominent in kidney in 2-day-old rats, is detected in lung and in epithelia of several tissues, but is not found in liver tissue. Retinal dehydrogenase activity peaks in kidney at Day 2 after birth and decreases gradually until adulthood, correlating well with RALDH expression. Weaker activity is also detectable in lungs but not in liver. Notably, distribution patterns of RALDH in kidney tissues are dramatically altered during postnatal development (P). From P0 to P6, hybridization is essentially concentrated within the marginal nephrogenic zone of the cortex. Expression progresses to deeper cortical layers from P12 to P16 and is intense in the medulla at P42, and focal expression is still detectable in the cortex. Immunocytochemical localization of RALDH in neonatal kidney shows staining mostly in cortical zone convoluted tubules and in adult rat shows staining in segments of distal and proximal tubules. These data suggest an important role for RALDH in modulating retinoic acid levels in different cell types during rat kidney development. The changing patterns of RALDH expression mirror stages of nephron formation in the developing rat kidney, strongly suggesting a central role for RALDH and thus for retinoids in controlling kidney development.
A New Quantitative Film Autoradiographic Method of Quantifying mRNA Transcripts for In Situ Hybridization
Authors: Palfi Arpad , Hatvani Laszlo , Gulya Karoly
Published: April 2011

We developed and tested a novel quantitative method for the quantification of film autoradiographs, involving a mathematical model and a dot-blot-based membrane standard scale. The exponential model introduced here, ROD = p1(1 - exp[ p2 x]), appropriately ( r2<0.999), describes the relation between relative optical density (ROD) and radioactivity ( x) in the range between 0 and 240 gray scale values (using a 256-gray scale level digitizer). By means of this model, standard curves with distinct quenching properties can be exactly interconverted, permitting the tissue-equivalent calibration of different standard scales. The membrane standard scale employed here has several advantages, including the flexible radioactivity range, the facile and rapid preparation technique, and the compact size. The feasibility of the quantification procedure is exemplified by the comparative quantification of multiple calmodulin mRNAs in the rat brain by in situ hybridization with [35S]-cRNA probes. The procedure for quantification provides a significant improvement in that the direct and exact comparison of radiolabeled species, even from different experiments, can be reliably performed. Further, the procedure can be adapted to the quantification of autoradiographs produced by other methods.
Effect of 3-Methylcholanthrene Administration on Expression of Cytochrome P-450 Isoforms Induced by Phenobarbital in Rat Hepatocytes
Authors: Mino Kazuto , Watanabe Jun , Kanamura Shinsuke
Published: April 2011

The effects of an inducer on expression of cytochrome P-450 (P-450) isoforms induced antecedently by another inducer are unknown. Thus, we examined the amount of phenobarbital (PB)-inducible P-450 isoforms (P-450 2B1/2B2) in hepatocytes from rats injected first with PB and then with 3-methylcholanthrene (MC) (PB+MC-treated animals) by quantitative immunohistochemistry. In addition, expression of P-450 2B2 mRNA was examined by in situ hybridization. In PB-treated animals, P-450 2B1/2B2 content increased in perivenular and midzonal hepatocytes. In PB+MC-treated animals, however, the PB-induced increase in 2B1/2B2 content was suppressed in perivenular hepatocytes but promoted in midzonal hepatocytes. The hybridization signal for P-450 2B2 mRNA appeared almost exclusively in perivenular hepatocytes after 24 hr of PB injection and disappeared after 48 hr of injection. In PB+MC-treated animals, however, strong hybridization signal was observed in midzonal and perivenular hepatocytes after 48 hr of PB injection. The promotion of the increase in P-450 2B1/2B2 content in midzonal hepatocytes in PB+MC-treated animals probably corresponds to the strong hybridization signal, whereas there appeared to be a divergence between the intensity of the signal and the content in perivenular hepatocytes. The results indicate that MC administration drastically influences the pattern of expression of P-450 isoforms induced by PB in perivenular and midzonal hepatocytes.
Pre-apoptotic Alterations in Hepatocytes of TNF α-treated Galactosamine-sensitized Mice
Authors: Angermüller Sabine , Künstle Gerald , Tiegs Gisa
Published: April 2011

Tumor necrosis factor (TNF) induces apoptotic death of hepatocytes in the galactosamine (GalN)-sensitized mouse liver after 5 hr. In our study, the most remarkable sign of the early stage of apoptosis was the focal rupture of the outer mitochondrial membrane. Parts of the inner membrane extended through the gap of the outer membrane, whereas the rest of the inner membrane still formed the cristae. This feature appeared in hepatocytes before chromatin condensation. With the diaminobenzidine technique for localization of cytochrome oxidase activity, the reaction product was detectable by light and electron microscopy. Ten percent of the hepatocytes were apoptotic, with condensed chromatin and high enzyme activity, 37% were pre-apoptotic, without chromatin condensation but high enzyme activity, and 53% had neither condensed chromatin nor a remarkable reaction product of cytochrome oxidase activity. Fas (APO-1, CD95) molecules on the plasma membrane of hepatocytes increased and were represented immunohistochemically in cells without chromatin condensation. DNA strand breaks were also detectable before chromatin aggregation. The results of this study indicate that mitochondria play a pivotal role in pre-apoptotic hepatocytes, together with an increase of the Fas molecule on the plasma membrane and with the occurrence of DNA strand breaks in the nucleus.
Identification of N- and O-linked Oligosaccharides in the Human Epididymis
Authors: Arenas María I. , Madrid Juan F. , Bethencourt Fermín R. , Fraile Benito , Paniagua Ricardo
Published: April 2011

The oligosaccharide sequences of glycoconjugates in the human normal epididymis and the nature of linkages were studied with lectin histochemistry. The usual terminal sequences of oligosaccharide side chains in epithelial cell secretions were Neu5Ac2,3Galβ 1,3GalNAc; SO4 Galβ1,3GalNAc; and Galβ1,4GlcNAc, and they were mainly found in O-linked glycoproteins. The lectin pattern of mitochondria-rich cells differed from that of principal cells.
Cortactin Localization in Actin-containing Adult and Fetal tissues
Authors: Wu Hong , Montone Kathleen T.
Published: April 2011

Cortactin is a tyrosine kinase substrate that binds to filamentous actin. It represents a highly conserved family of perimembrane signaling proteins. The human homologue of cortactin is encoded by gene EMS1, which is amplified in some human breast, head, and neck carcinomas. This study shows that cortactin is preferentially localized to the apical surfaces of the polarized epithelium, specifically, to the terminal web of small bowel epithelium and to apical surfaces of the proximal renal tubules, thyroid follicles, and bronchiolar epithelium. Cortactin is also seen in cell and tissue types with actin-based contractile capacities, including smooth and striated muscle and myoepithelium.
Choline Acetyltransferase (ChAT) Immunoreactivity in Paraffin Sections of Normal and Diseased Intestines
Authors: Ratcliffe Elyanne M. , deSa Derek J. , Dixon Michael F. , Stead Ronald H.
Published: April 2011

There is increasing interest in localizing nerves in the intestine, especially specific populations of nerves. At present, the usual histochemical marker for cholinergic nerves in tissue sections is acetylcholinesterase activity. However, such techniques are applicable only to frozen sections and have uncertain specificity. Choline acetyltransferase (ChAT) is also present in cholinergic nerves, and we therefore aimed to establish a paraffin section immunocytochemical technique using an anti-ChAT antibody. Monoclonal anti-choline acetyltransferase (1.B3.9B3) and a biotin-streptavidin detection system were used to study the distribution of ChAT immunoreactivity (ChAT IR) in paraffin-embedded normal and diseased gastrointestinal tracts from both rats and humans. Optimal staining was seen after 6-24 hr of fixation in neutral buffered formalin and overnight incubation in 1 μg/ml of 1.B3.9B3, with a similar distribution to that seen in frozen sections. In the rat diaphragm (used as a positive control), axons and motor endplates were ChAT IR. Proportions of ganglion cells and nerve fibers in the intramural plexi of both human and rat gastrointestinal tracts were also ChAT IR, as well as extrinsic nerve bundles in aganglionic segments of Hirschsprung's disease. Mucosal cholinergic nerves, however, were not visualized. In addition, non-neuronal cells such as endothelium, epithelium, and inflammatory cells were ChAT IR. We were able to localize ChAT to nerves in formalin-fixed, paraffin-embedded sections. The presence of ChAT IR in non-neuronal cells indicates that this method should be used in conjunction with other antibodies. Nevertheless, it proves to be a useful technique for studying cholinergic neuronal distinction in normal tissues and pathological disorders.
Immunohistochemical Analysis of the Distribution of the Human ATPase (hASNA-I) in Normal Tissues and Its Overexpression in Breast Adenomas and Carcinomas
Authors: Kurdi-Haidar Buran , Heath Dennis , Naredi Peter , Varki Nissi , Howell Steven B.
Published: April 2011

Human ATPase (hASNA-I) is a novel human gene recently cloned on the basis of homology to the arsA gene of bacteria. Its protein product is an ATPase that is free in the cytoplasm and bound in the perinuclear area and nucleolus in human cells. We prepared the hASNA-I-specific 5G8 monoclonal antibody and used it to investigate the expression of hASNA-I in normal human tissues and breast cancers. hASNA-I was detected immunohistochemically only in the epithelial cells of the liver, kidney, and stomach wall, in the adrenal medulla, in the islet cells of the pancreas, in the red pulp of the spleen, and in cardiac and skeletal muscle. No staining was observed in the uterus, testis, lung, thyroid, cerebellum, and large intestine. Although no staining was also observed in normal breast tissue, all four cases of breast fibroadenomas and all 15 cases of either primary or metastatic breast carcinoma demonstrated increased staining. No embryological or functional common denominator is readily apparent. However, the increased expression in malignant breast cells is of particular interest with respect to the use of this antibody for screening of cytological specimens.
Deimination of 70-kD Nuclear Protein During Epidermal Apoptotic Events In Vitro
Authors: Mizoguchi Masayuki , Manabe Motomu , Kawamura Yasushi , Kondo Yukiko , Ishidoh Kazumi , Kominami Eiki , Watanabe Kazutaka , Asaga Hiroaki , Senshu Tatsuo , Ogawa Hideoki
Published: April 2011

Peptidylarginine deiminase (PAD) is the enzyme responsible for converting protein-bound arginine residues to citrulline. It has recently been shown that a number of epidermal proteins, including filaggrin, trichohyalin, and keratins, are deiminated by the action of PAD, suggesting a possible role for protein deimination during the final stages of epidermal differentiation. We report here a novel PAD substrate found during the course of identifying deiminated proteins in cultured rat epidermal keratinocytes. We found that a 70-kD protein localized to the periphery of the nucleus was preferentially deiminated after ionomycin treatment in the presence of 2 mM calcium and was associated with apoptotic events in these cells. Furthermore, we discovered that the deimination of nuclear protein could be induced by transfection of a PAD cDNA into rat epidermal keratinocytes. These data suggest that PAD may act on the 70-kD nuclear protein to induce disassembly of the nuclear lamina and promote apoptosis during terminal epidermal differentiation.
Expression of 1,25-Dihydroxy Vitamin D3 Receptor in Breast Carcinoma
Authors: Friedrich Michael , Rafi Leyla , Schmidt Werner , Tilgen Wolfgang , Reichrath Jörg
Published: April 2011

We investigated immunohistochemically the expression of 1,25 dihydroxy vitamin D3 receptors (VDRs) in normal human breast tissue and in breast carcinomas. For the first time, a VDR immunoreactivity score (VDR-IRS) in breast tissue is presented. Mean VDRIRS in breast carcinomas was 7.28 compared to 1.55 in normal breast tissue. Comparing staining patterns for VDR and Ki-67, no visual correlation was found, indicating that VDR upregulation in breast carcinomas is not exclusively controlled by the proliferative activity of these tumor cells. Our study adds to the body of evidence that breast tissue may be a sensitive target organ for therapeutically applied new vitamin D analogues that exert few calcemic side effects.
Immunohistochemical Localization of Carboxypeptidases E and D in the Human Placenta and Umbilical Cord
Authors: Reznik Sandra E. , Salafia Carolyn M. , Lage Janice M. , Fricker Lloyd D.
Published: April 2011

Carboxypeptidase E (CPE) is highly concentrated in neuroendocrine tissues and is the only carboxypeptidase detected in mature secretory vesicles. Carboxypeptidase D (CPD), a carboxypeptidase with CPE-like activity, is widely distributed in tissues and is present in the trans-Golgi network. Previous work had shown that both CPE and CPD are expressed in the human placenta and that CPD is expressed at much higher levels than CPE. The present work provides evidence for the co-localization of CPE and CPD to basal plate extravillous trophoblasts and maternal uteroplacental vascular endothelial cells, chorionic villous endothelial cells, amnionic epithelial cells, and umbilical venous and arterial smooth muscle cells. Whereas the intensity of CPD immunostaining is similar in the placenta and umbilical cord, CPE staining in the placenta is much weaker than in the umbilical cord, suggesting that CPD plays a more important role in the processing of placental peptides. Immunoelectron microscopy of umbilical venous smooth muscle cells shows subcellular localization of both enzymes to the rough endoplasmic reticulum. In addition, CPE is present just subjacent to the cell membrane. The difference in cellular and subcellular localization between the two enzymes indicates that they perform distinct functions in the processing of placental peptides and proteins.
Widespread Expression of Serum Amyloid A in Histologically Normal Human Tissues: Predominant Localization to the Epithelium
Authors: Urieli-Shoval Simcha , Cohen Patrizia , Eisenberg Shlomit , Matzner Yaacov
Published: April 2011

Serum amyloid A (SAA) is an acute-phase reactant whose level in the blood is elevated to 1000-fold as part of the body's responses to various injuries, including trauma, infection, inflammation, and neoplasia. As an acute-phase reactant, the liver has been considered to be the primary site of expression. However, limited extrahepatic SAA expression was described in mouse tissues and in cells of human atherosclerotic lesions. Here we describe nonradioactive in situ hybridization experiments revealing that the SAA mRNA is widely expressed in many histologically normal human tissues. Expression was localized predominantly to the epithelial components of a variety of tissues, including breast, stomach, small and large intestine, prostate, lung, pancreas, kidney, tonsil, thyroid, pituitary, placenta, skin epidermis, and brain neurons. Expression was also observed in lymphocytes, plasma cells, and endothelial cells. RT-PCR analysis of selected tissues revealed expression of the SAA1, SAA2, and SAA4 genes but not of SAA3, consistent with expression of these genes in the liver. Immunohistochemical staining revealed SAA protein expression that colocalized with SAA mRNA expression. These data indicate local production of the SAA proteins in histologically normal human extrahepatic tissues.
Specificity of Antibodies to Nitric Oxide Synthase Isoforms in Human, Guinea Pig, Rat, and Mouse Tissues
Authors: Coers Wilko , Timens Wim , Kempinga Claudia , Klok Pieter A. , Moshage Han
Published: April 2011

Ten commercially available rabbit polyclonal anti-NOS antibodies were tested for their immunohistological applicability in normal human, guinea pig, rat, and mouse organs. Most antibodies reacted as expected and described in the literature with various tissues of the investigated species. Several antibodies did not react with the expected cell populations in a certain species, or reacted in previously unknown patterns. In addition, different antibodies to the same isoform rarely detected identical cell populations, even within one species. Most of these unexpected immunoreactivities were observed in bronchial epithelial, glomerular epithelial, and vascular smooth muscle cells. These unexpected results usually occurred when the antibodies were tested in other organs or species than that to which they were originally raised. We therefore strongly recommend the use of anti-NOS antibodies only after careful immunohistological and biochemical analysis of their reactivity in the organ and species to be studied.
Distinct Patterns of NCAM Expression Are Associated with Defined Stages of Murine Hair Follicle Morphogenesis and Regression
Authors: Müller-Röver Sven , Peters Eva J. M. , Botchkarev Vladimir A. , Panteleyev Andrei , Paus Ralf
Published: April 2011

Hair follicle development, growth (anagen), and regression (catagen) largely result from bidirectional epithelial-mesenchymal interactions whose molecular basis is still unclear. Because adhesion molecules are critically involved in pattern formation and because the fundamental importance of neural cell adhesion molecule (NCAM) for feather development has been demonstrated, we studied the protein expression patterns of NCAM during hair follicle development and regression in the C57BL/6 mouse model. During murine hair follicle development, NCAM immunoreactivity (IR) was first detected on epithelial hair placodes and later on selected keratinocytes in the distal outer root sheath. Mesenchymal NCAM immunoreactivity (IR) was noted on fibroblasts of the future dermal papilla (DP) and the perifollicular connective tissue sheath. Fetal hair follicle elongation coincided with strong, ubiquitous dermal NCAM IR, which remained strong until the follicles entered into their first neonatal catagen. At this time, the strong interfollicular dermal NCAM IR decreased substantially. During consecutive hair cycles, mesenchymal NCAM IR was seen exclusively on DP and perifollicular connective tissue sheath fibroblasts and on the trailing cells of regressing catagen hair follicles. These highly restricted and developmentally controlled expression patterns suggest an important role for NCAM in hair follicle topobiology during morphogenesis and cyclic remodeling of this miniorgan.
Ultrastructural Aspects of the DNA Polymerase α Distribution During the Cell Cycle
Authors: Lattanzi Giovanna , Galanzi Angela , Gobbi Pietro , Falconi Mirella , Matteucci Alessandro , Breschi Lorenzo , Vitale Marco , Mazzotti Giovanni
Published: April 2011

We studied the nuclear topography of the replicating enzyme DNA polymerase α in HeLa cells by transmission electron microscopy and field emission in lens scanning electron microscopy. Cells were synchronized at the G1/S-phase boundary and samples of the different phases of the cell cycle were labeled with an anti-DNA polymerase α antibody detected by an immunogold reaction. DNA synthesis was detected by immunogold labeling after bromodeoxyuridine administration. The typical labeling pattern of DNA polymerase α observed in G1 - and S-phase cells was represented by circular structures 80-100 nm in diameter surrounding an electron-dense area. In double labeled samples these circular structures were associated with bromodeoxyuridine-containing DNA replication sites, forming rosette-like structures. Field emission scanning electron microscopy performed on ultrathin cryosections revealed the chromatin fibers underlying DNA polymerase α complexes and showed that the size of the rosette-like structures corresponded to the diameter of chromatin foldings. G2- and M-phase cells showed a spread distribution of DNA polymerase α. The evidence of DNA polymerase α circular arrangement exclusively in G1- and S-phase cells, obtained by such different approaches, allowed us to consider the three-dimensional structures as DNA replication areas.
Chondrocyte Differentiation in a Rat Mesenchymal Cell Line
Authors: Lunstrum Gregory P. , Keene Douglas R. , Weksler Nicole B. , Cho Yoon-Jae , Cornwall Marcus , Horton William A.
Published: April 2011

We used a combination of morphologic and histochemical methods to demonstrate that rat calvaria-derived mesenchymal cells, RCJ 3.1C5.18, in culture progress through the differentiation pathway exhibited by chondrocytes in the endochondral growth plate. The cells were grown either as monolayer or suspension cultures. Subconfluent monolayer cultures did not express markers typical of chondrocyte phenotypes. However, after reaching confluency the cells formed nodules of chondrocytic cells separated by cartilage-appearing matrix and encapsulated by fibroblast-like cells. Suspension culture produced cell aggregates with similar characteristics. Matrix in both the nodules and aggregates stained for collagen Types II and XI and aggrecan, and some cells displayed a distinctive pericellular matrix that stained for Type X collagen. Mineralization was evident in older cultures. By electron microscopy, most cells in the aggregates appeared as typical chondrocytes. However, some larger cells were surrounded by a “mat” of matrix comprised of hexagonal arrays of dense nodules interconnected by a filamentous network. Immunogold localization confirmed the presence of collagen Type X in this matrix. Analysis of markers of chondrocyte differentiation and terminal differentiation over time showed that these markers were acquired sequentially over 2 weeks of culture. This model system will be useful to study the regulation of various steps in the chondrocyte differentiation pathway.
Ultrastructural Localization of β-Actin and Amphoterin mRNA in Cultured Cells: Application of Tyramide Signal Amplification and Comparison of Detection Methods
Authors: Punnonen Eeva-Liisa , Fages Carole , Wartiovaara Jorma , Rauvala Heikki
Published: April 2011

We describe a nonradioactive preembedding in situ hybridization protocol using digoxigenin-labeled RNA probes and tyramide signal amplification to increase the sensitivity of detection. The protocol is sensitive enough for electron microscopic localization of endogenous messenger RNAs encoding β-actin and amphoterin. Three visualization methods were compared: diaminobenzidine enhanced by nickel, Nanogold enhanced by silver and gold toning, and fluorescently labeled tyramides. Diaminobenzidine and Nanogold can be used in both light and electron microscopy. The nickel-enhanced diaminobenzidine was the most sensitive visualization method. It is easy to accomplish but a drawback is poor spatial resolution, which restricts its use at high magnifications. Nanogold visualization has considerably better spatial resolution and is therefore recommended for electron microscopy. Fluorescent tyramides, especially TRITC-tyramide, offer a good detection method for fluorescence and confocal microscopy. The methods were used to localize amphoterin and β-actin mRNAs in motile cells. Both mRNAs were found in the soma and cell processes. In double labeling experiments, β-actin mRNA localized to filamentous structures that also contained ribosomal proteins. Especially in the cortical cytoplasm, β-actin mRNA was associated with actin filaments. Direct localization to microtubules was only rarely seen.
Presence in Human Skeletal Muscle of an AMP Deaminase-associated Protein That Reacts with an Antibody to Human Plasma Histidine-Proline-rich Glycoprotein
Authors: Sabbatini Antonietta R. M. , Ranieri-Raggi Maria , Pollina Luca , Viacava Paolo , Ashby John R. , Moir Arthur J. G. , Raggi Antonio
Published: April 2011

Histidine-proline-rich glycoprotein (HPRG) is a protein that is synthesized by parenchimal liver cells. The protein has been implicated in a number of plasma-specific processes, including blood coagulation and fibrinolysis. We have recently reported the association of an HPRG-like protein with rabbit skeletal muscle AMP deaminase (AMPD). The results of the immunological analysis reported here demonstrate that an antibody against human plasma HPRG reacts with an AMPD preparation from human skeletal muscle. To probe the localization of the putative HPRG-like protein in human skeletal muscle, serial sections from frozen biopsy specimens were processed for immunohistochemical and histoenzymatic stains. A selective binding of the anti-HPRG antibody to Type IIB muscle fibers was detected, suggesting a preferential association of the novel protein to the AMPD isoenzyme contained in the fast-twitch glycolytic fibers.
A Method for Counterstaining Tissues in Conjunction with the Glyoxylic Acid Condensation Reaction for Detection of Biogenic Amines
Authors: Guidry Guy
Published: April 2011

A method is described for counterstaining tissue for use with the glyoxylic acid reaction for visual detection of biogenic amines. Counterstaining is achieved by addition of the fluorescent dye malachite green to the sucrose-phosphate-glyoxylic acid (SPG) solution used for processing of cryostat sections of unfixed tissues. When bound to tissues, the dye provides red-orange fluorescence of background tissue, which contrasts well with the green to yellow fluorescence induced by the glyoxylic acid reaction product formed with biogenic amines. The counterstaining technique is demonstrated in a number of catecholamine-containing peripheral tissues and is compared to sections that were processed without counterstaining.
Three Distinct Classes of the α-Subunit of the Nuclear Pore-targeting Complex (Importin-α) Are Differentially Expressed in Adult Mouse Tissues
Authors: Kamei Yasuhiro , Yuba Shunsuke , Nakayama Tatsuo , Yoneda Yoshihiro
Published: April 2011

The process of active nuclear protein transport is mediated by the nuclear localization signal (NLS). An NLS-containing karyophile forms a stable complex, termed the nuclear pore-targeting complex, to target nuclear pores. The α-subunit of the complex (importin-α) binds to the NLS and the β-subunit (importin-β-) carries the α-subunit, bound to the NLS substrate, into the nucleus. To date, five mouse α-subunits have been identified and classified into three subfamilies (α-P, α-Q, and α-S). The expression of these α-subunits and the β-subunit in various adult mouse tissues was examined by immunoblotting and immunohistochemistry using antibodies specific for each subfamily of the α-subunit or the β-subunit. The β-subunit was found to be ubiquitously expressed, whereas each subfamily of the α-subunit showed a unique expression pattern in various tissues, especially in brain and testis. In brain, the expression of α-P was not observed, whereas α-S was significantly expressed in Purkinje cells, and pyramidal cells of the hippocampus and cerebral cortex. In testis, α-P was expressed predominantly in primary spermatocytes, whereas α-Q was found mainly in Leydig cells. Expression of α-S was detected in almost all cells in convoluted seminiferous tubules and Leydig cells to a similar extent. These results suggest that nuclear protein import may be controlled in a tissue-specific manner by α-subunit family proteins.
Visualization of Cell Surface Vasopressin V1a Receptors in Rat Hepatocytes with a Fluorescent Linear Antagonist
Authors: Tran Dien , Durroux Thierry , Stelly Nicole , Seyer René , Tordjmann Thierry , Combettes Laurent , Claret Michel
Published: April 2011

To visualize cell surface V1a vasopressin receptors in rat hepatocytes in the absence of receptor-mediated endocytosis, we used a high-affinity fluorescent linear antagonist, Rhm8 -PVA. Epifluorescence microscopy (3CCD camera) and fluorescence spectroscopy were used. Rhm8 -PVA alone did not stimulate Ca2+ signals and competitively blocked Ca2+ signals (Kinact of 3.0 nM) evoked by arginine vasopressin (vasopressin). When rat hepatocytes were incubated with 10 nM of Rhm8 -PVA for 30 min at 4C, the fluorescent antagonist bound to the surface of cells, presumably the plasma membrane. The V1a receptor specificity of Rhm8 -PVA binding was confirmed by its displacement by the nonfluorescent antagonist V4253 and by the natural hormone vasopressin at 4C. Prior vasopressin-mediated endocytosis of V1a receptors at 37C abolished binding of the labeled antagonist, whereas in non-preincubated cells, Rhm8 -PVA labeled the cell surface of rat hepatocytes. When cells labeled with Rhm8 -PVA at 4C were warmed to 37C to initiate receptor-mediated internalization of the fluorescent complex, Rhm8 -PVA remained at the cell surface. Incubation temperature at 4C or 37C had little effect on binding of Rhm8 -PVA. We conclude that Rhm8 -PVA is unable to evoke receptor-mediated endocytosis and can readily be used to visualize cell surface receptors in living cells.
Specific Localization of Membrane Dipeptidase and Dipeptidyl Peptidase IV in Secretion Granules of Two Different Pancreatic Islet Cells
Authors: Grondin Gilles , Hooper Nigel M. , LeBel Denis
Published: April 2011

Endocrine cells require several protein convertases to process the precursors of hormonal peptides that they secrete. In addition to the convertases, which have a crucial role in the maturation of prohormones, many other proteases are present in endocrine cells, the roles of which are less well established. Two of these proteases, dipeptidyl peptidase IV (EC 3.4.14.5) and membrane dipeptidase (EC 3.4.13.19), have been immunocytochemically localized in the endocrine pancreas of the pig. Membrane dipeptidase was present exclusively in cells of the islet of Langerhans that were positive for the pancreatic polypeptide, whereas dipeptidyl peptidase IV was restricted to cells positive for glucagon. Both enzymes were observed in the content of secretory granules and therefore would be released into the interstitial space as the granules undergo exocytosis. At this location they could act on secretions of other islet cells. The relative concentration of dipeptidyl peptidase IV was lower in dense glucagon granules, where the immunoreactivity to glucagon was higher, and vice versa for light granules. This suggests that, in A-cells, dipeptidyl peptidase IV could be sent for degradation in the endosomal/lysosomal compartment during the process of granule maturation or could be removed from granules for continuous release into the interstitial space. The intense proteolytic activity that takes place in the endocrine pancreas could produce many potential dipeptide substrates for membrane dipeptidase.
Electron Microscopic In Situ DNA Nick End-labeling in Combination with Immunoelectron Microscopy
Authors: Ishida-Yamamoto Akemi , Yamauchi Toshihiro , Tanaka Hikaru , Nakane Hiroshi , Takahashi Hidetoshi , lizuka Hajime
Published: April 2011

We describe an in situ DNA nick end-labeling method that can be performed at the electron microscopic level and can also be combined with immunoelectron microscopy. As the materials, we used skin tissues from normal skin and from Bowen's disease that had been cryofixed, freeze-substituted, and embedded in Lowicryl K11M resin. Ultrathin sections were cut and incubated with a reaction buffer containing digoxigenindUTP and terminal deoxynucleotidyl transferase. Digoxigenin nucleotides were labeled with anti-digoxigenin antibodies conjugated with colloidal gold. Specific signals were detected in the condensed chromatin of differentiated epidermal cells and hair follicles in normal skin and of dyskeratotic cells in Bowen's disease. The labeling density over chromosomal areas of apoptotic cells was significantly higher than that over chromosomal areas of mitotic cells or cytoplasmic areas. Ultrastructure was well preserved and double staining with an anti-keratin antibody was also successfully performed. This simple method has a wide range of applications to identify the nature of apoptotic cells and explore the mechanisms of apoptosis.
Expression of Embryonic Fibronectin Isoform EIIIA Parallels α-Smooth Muscle Actin in Maturing and Diseased Kidney
Authors: Barnes Veronique L. , Musa John , Mitchell Ronda J. , Barnes Jeffrey L.
Published: April 2011

In this study we examined if an association exists between expression of an alternatively spliced “embryonic” fibronectin isoform EIIIA (Fn-EIIIA) and α-smooth muscle actin (α-SMA) in the maturing and adult rat kidney and in two unrelated models of glomerular disease, passive accelerated anti-glomerular basement membrane (GBM) nephritis and Habu venom (HV)-induced proliferative glomerulonephritis, using immunohistochemistry and in situ hybridization. Fn-EIIIA and α-SMA proteins were abundantly expressed in mesangium and in periglomerular and peritubular interstitium of 20-day embryonic and 7-day (D-7) postnatal kidneys in regions of tubule and glomerular development. Staining was markedly reduced in these structures in maturing juvenile (D-14) kidney and was largely lost in adult kidney. Expression of Fn-EIIIA and α-SMA was reinitiated in the mesangium and the periglomerular and peritubular interstitium in both models and was also observed in glomerular crescents in anti-GBM nephritis. Increased expression of Fn-EIIIA mRNA by in situ hybridization corresponded to the localization of protein staining. Dual labeling experiments verified co-localization of Fn-EIIIA and α-SMA, showing a strong correlation of staining between location and staining intensity during kidney development, maturation, and disease. Expression of EIIIA mRNA corresponded to protein expression in developing and diseased kidneys and was lost in adult kidney. These studies show a recapitulation of the co-expression of Fn-EIIIA and α-SMA in anti-GBM disease and suggest a functional link for these two proteins.
Expression of the MUC 6 Mucin Gene in Development of the Human Kidney and Male Genital Ducts
Authors: Reid Colm J. , Harris Ann
Published: April 2011

The MUC 6 mucin cDNA was isolated from a human stomach cDNA library and has been shown to be expressed in a number of other tissues in the gastrointestinal tract, including the gallbladder, pancreas, and parts of the ileum and colon. Here we establish that MUC 6 is expressed transiently in the nephrogenic zone of the kidney in the early mid-trimester of development. MUC 6 transcripts were detected in the epithelium of ureteric buds at 13 weeks and at lower levels from 17 to 23 weeks of gestation. Traces of MUC 6 mRNA were seen in the collecting ducts but not elsewhere in the developing kidney, and MUC 6 glycoprotein was detected in the epithelium of ureteric buds and collecting ducts. MUC 6 transcripts were absent from adult kidney. This pattern of expression of MUC 6 in the developing kidney suggests a role in epithelial organogenesis. MUC 6 transcripts were also present at low levels in mid-trimester epididymal epithelium.
Immunohistochemical Localization of EphA5 in the Adult Human Central Nervous System
Authors: Olivieri Gianfranco , Miescher Guido C.
Published: April 2011

To better understand the functional role of EphA5 in the adult human central nervous system (CNS), we performed an immunohistochemical mapping study. EphA5, like other members of the Elk/Eph family of receptor tyrosine kinases, was widely distributed in CNS neurons. However, the distribution of the neuronal staining was not uniform. The abundance of stained neurons appeared to increase from the forebrain to the hindbrain and spinal cord. Glial and endothelial tissue was unstained. These findings are consistent with the existence of receptor and ligand gradients in different brain regions. The localization of EphA5 to motor and sensory neurons is consistent with a role of EphA5 in neural plasticity, cell-cell recognition, and topographical orientation of neuronal systems.
Immunocytochemical Localization of Phospholipase C Isozymes in Cord Blood and Adult T-lymphocytes
Authors: Miscia Sebastiano , Baldassarre Angela Di , Cataldi Amelia , Rana Rosa Alba , Pietro Roberta Di , Bosco Domenico , Grilli Alfredo , Amerio Giulia , Sabatino Giuseppe
Published: April 2011

The response of T-cells to peptide antigen plus major histocompatibility complex (MHC) consists of a series of cellular events collectively called T-cell activation. An essential component of this pathway is phospholipase C (PLC)γ 1, whose hydrolytic activity increases rapidly after binding of ligands to the T-cell receptor (TCR) and consequent activation of tyrosine kinases. Recent studies also suggest a GTP binding protein-dependent activation of PLCβ during the early steps of T-cell activation. On the basis of these findings, we first checked the expression of PLC isoforms by Western blotting and by confocal and electron microscopy techniques, and then we looked for the phosphoinositide breakdown induced by CD3 engagement in cord and adult T-lymphocytes. Our results indicated that PLCβ1 was almost exclusively expressed in cord T-cells, whereas PLC β2 was more strongly represented in the adult. The amount of PLCγ 1 was found to be larger in the adult than in cord cells. No significant differences were found in PLCγ 2 and $2 expression. PLCγ 1 was scarcely detectable. On CD3 stimulation, adult lymphocytes gave rise, as expected, to a dramatic increase in phosphoinositide breakdown, whereas in cord cells this response was scarcely detected. These results indicate that a shift in PLC expression occurs in the postnatal period and that this change is associated with induction of the capability to respond to CD3 engagement with phosphoinositide hydrolysis.
Detection of Pathological Zinc Accumulation In Neurons: Methods for Autopsy, Biopsy, and Cultured Tissue
Authors: Suh Sang Won , Listiack Kathy , Bell Brent , Chen Jefferson , Motamedi Massoud , Silva David , Danscher Gorm , Whetsell William , Thompson Richard , Frederickson Christopher
Published: April 2011

It has been repeatedly shown that synaptically released zinc contributes to excitotoxic neuronal injury in ischemia, epilepsy, and mechanical head trauma. Such zinc-induced injury leaves an unmistakable “footprint” in the injured neurons, allowing an easy and unambiguous postmortem diagnosis. This footprint is the presence of weakly bound, histochemically reactive zinc in the cytoplasm of the perikaryon and proximal dendrites. Such staining appears to be a necessary and sufficient marker for zinc-induced neuronal injury. Here we show how to prepare and stain tissue from biopsy, autopsy, or experimental animal sources for maximal contrast and visibility of zinc-injured neurons.
Pretreatment in a High-pressure Microwave Processor for MIB-1 Immunostaining of Cytological Smears and Paraffin Tissue Sections to Visualize the Various Phases of the Mitotic Cycle
Authors: Suurmeijer Albert J.H. , Boon Mathilde E.
Published: April 2011

In many pathology laboratories, both microwave ovens and pressure cookers are used for pretreatment of cytologic smears and paraffin sections to allow MIB-1 staining. For both methods there are two problems. First, the results cannot be used for quantitation because standardization is impossible. Second, the staining results are often suboptimal, resulting in negative staining of cells in the G1- and S-phases. When pretreatment is performed in a microwave processor, allowing microwave heating under pressure, precise temperature monitoring becomes possible. In addition, the importance of the pH of the buffer was studied using a test battery series. Optimal staining is achieved at a temperature of 115C, 10 min, pH 6. This method proved to be highly reproducible. Because the immunostaining results are optimal, the various phases of the cell cycle can be defined in the sections and smears. In addition, the perinucleolar staining of the late G1-phase is optimally visualized and nuclei of the stable pKi-67 pathway can be identified. Under suboptimal conditions, in particular, the number of cells in the late G1-phase are underestimated in the MIB-1 counts. (J Histochem Cytochem 47:1015–1020, 1999)
Quantitative Immunocytochemical Study of Blood-Brain Barrier Glucose Transporter (GLUT-1) in Four Regions of Mouse Brain
Authors: Dobrogowska Danuta H. , Vorbrodt Andrzej W.
Published: April 2011

Application of immunogold cytochemistry revealed polar (asymmetric) distribution of GLUT-1 in mouse brain microvascular endothelia, representing the anatomic site of the blood-brain barrier (BBB). This polarity was manifested by an approximately threefold higher immunolabeling density of the abluminal than the luminal plasma membrane of the endothelial cells. The immunoreaction for GLUT-1 in nonbarrier continuous (skeletal muscle) or fenestrated (brain circumventricular organs) microvascular endothelial cells was absent. In the choroid plexus, the basolateral plasmalemma of the epithelial cells was labeled more intensely than the vascular fenestrated endothelium. Addition of morphometry to the applied immunogold technique makes it possible for even subtle differences to be revealed in the density of immunolabeling for GLUT-1 in blood microvessels located in four brain regions. We found that the density of immunosignals in the microvessels supplying the cerebral cortex, hippocampus, and cerebellum was essentially similar, whereas in the olfactory bulb it was significantly lower. Asymmetric distribution of GLUT-1 in the endothelial plasma membranes presumably leads to a reduced concentration of glucose molecules in the endothelial cells compared to blood plasma and also secures their more rapid transport across the abluminal plasmalemma to the brain parenchyma. (J Histochem Cytochem 47:1021–1029, 1999)
Immunohistochemical Staining Patterns of MUC1, MUC2, MUC4, and MUC5AC Mucins in Hyperplastic Polyps, Serrated Adenomas, and Traditional Adenomas of the Colorectum
Authors: Biemer-Hüttmann Anne-Eve , Walsh Michael D. , McGuckin Michael A. , Ajioka Yoichi , Watanabe Hidenobu , Leggett Barbara A. , Jass Jeremy R.
Published: April 2011

We studied the distribution of the four human apomucins MUC1, MUC2, MUC4, and MUC5AC in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum using immunohistochemical techniques, with the aim of comparing and contrasting their patterns of expression. A series of 12 hyperplastic polyps, 27 serrated adenomas, and 20 traditional adenomas was studied. No significant change in apomucin expression was observed in traditional adenomas compared with normal colorectal epithelium, except for MUC5AC, which was present in 12 of the adenomas (60%) and only 20% of the normal samples. In both hyperplastic polyps and serrated adenomas, MUC2 and MUC5AC mucin expression was consistently and markedly increased. In 50% of the hyper-plastic polyps, MUC4 was reduced but in the remaining cases was similar to normal. Loss of MUC4 expression was observed in all serrated adenomas. MUC1 was not increased in the hyperplastic polyps but increased expression was seen in 17 of the serrated adenomas (63%). Similar altered distribution patterns of MUC2, MUC4, and MUC5AC were seen in hyperplastic polyps and serrated adenomas, whereas traditional adenomas showed little change from normal patterns of expression. Although hyperplastic polyps are commonly defined as benign lesions without neoplastic potential, the similar phenotypes of hyper-plastic and serrated adenomas and the existence of mixed polyps suggest that these lesions may represent a histogenetic continuum. (J Histochem Cytochem 47:1039–1047, 1999)