bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative immunoelectron microscopic analysis of the localization and induction of 25-hydroxyvitamin D3 24-hydroxylase in rat kidney.
Authors: Iwata K , Yamamoto A , Satoh S , Ohyama Y , Tashiro Y , Setoguchi T
Published: April 2011

25-Hydroxyvitamin D3 24(R)-hydroxylase (24-hydroxylase) is involved in the metabolism and regulation of vitamin D3 and is markedly induced by administration of vitamin D3. We detected this enzyme by electron microscopy and an immunogold technique along nephrons of normal and vitamin D3-administered rats. After the rats were administered vitamin D3, 50,000 IU/day for 1 week, they were perfusion-fixed with a paraformaldehyde solution. The fixed kidneys were then removed and embedded in LR White resin. Ultrathin sections were prepared and labeled by the immunogold technique using a mouse anti-rat 25-hydroxyvitamin D3 24-hydroxylase monoclonal antibody. We counted the number of gold particles bound per micron 2 of the mitochondria (particle density) of the tubule epithelial cells along the nephrons. In normal and vitamin D3-administered rats, gold particles were observed in the mitochondria of epithelial cells along the tubules. In normal rats, gold labeling for 24-hydroxylase was statistically significant (p < 0.05), in the S1-S2 segments, the S3 segment of the proximal tubules, and in the distal convoluted tubules. In the rats administered vitamin D3, the particle density increased significantly (p < 0.05) by about 12-fold in the S1-S2 segments of the proximal tubules, whereas it increased less markedly in other parts of the nephron. The marked induction of the S1-S2 segments of the proximal tubules suggests that these segments play an important role in the regulation of vitamin D3 metabolism.
Anionic sites on Reissner's membrane, stria vascularis, and spiral prominence.
Authors: Torihara K , Morimitsu T , Suganuma T
Published: April 2011

We demonstrated anionic sites on the lateral wall of cochlear duct and Reissner's membrane (RM) of ICR mice by Lowicryl K4M resin post-embedding and poly-L-lysine-colloidal gold conjugate (PL-CG) as a polycationic probe. The basement membrane and endolymphatic cell surface of RM were labeled with PL-CG pH 2.5 and pH 1.0. However, the perilymphatic cell surface was not labeled. PL-CG pH 2.5 and pH 1.0 strongly labeled the endolymphatic surface of the spiral prominence epithelium (SP), whereas the endolymphatic surface of the marginal cell (MC) in the stria vascularis was not labeled. Pre-digestion with several glycosidases eliminated PL-CG labeling. Our result suggests that an anionic charge located on the basement membrane of RM is largely due to the presence of heparan sulfate, chondroitin sulfate, and hyaluronic acid. An anionic charge on the endolymphatic cell surface of RM was mainly dependent on the presence of heparan sulfate. An anionic charge on the SP epithelium was caused to a substantial degree by chondroitin sulfate. We obtained histochemical evidence that the glycoconjugate content of the MC surface was quite different from that of the endolymphatic cell surface of RM and SP. We also identified RM-MC and SP-MC junctions at the ends of the stria vascularis between the marginal cells and the other endolymphatic epithelial cells of the cochlear duct.
The use of electron microscopic immunocytochemistry with silver-enhanced 1.4-nm gold particles to localize GAD in the cerebellar nuclei.
Authors: Gilerovitch H G , Bishop G A , King J S , Burry R W
Published: April 2011

Silver enhancement of small gold particles can be used with pre-embedding immunocytochemistry to analyze the distribution of label over cell organelles. We have developed a method that improves tissue morphology, has good penetration of reagents, and allows greater control of silver enhancement of 1.4-nm gold. In this study we analyzed the distribution of glutamic acid decarboxylase (GAD), a synthetic enzyme for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), in the cerebellar nuclei of the mouse. Pre-embedding immunocytochemistry was carried out on brain sections fixed with high concentrations of glutaraldehyde and sodium metabisulfite. After incubations with a monoclonal antibody to GAD and a 1.4-nm NanoGold-labeled secondary antibody, sections were silver-enhanced with N-propyl gallate as a reducing agent and MES as a new buffer system. In the cerebellar nuclei, GAD label was specifically localized in axon terminals over clusters of synaptic vesicles. These terminals formed axosomatic and axodendritic contacts. The majority of GAD-labeled terminals had cytological characteristics indicating their origin from Purkinje cells, which are known to contain GAD.
Ultrastructural localization of ras-related proteins using epitope-tagged plasmids.
Authors: Robertson D , Paterson H F , Adamson P , Hall A , Monaghan P
Published: April 2011

To determine the ultrastructural distribution of H-ras, the rho proteins rho-A, rho-B, rho-C, and the rac1 protein (members of the ras GTP-binding protein family), we used cDNA expression plasmids in which a short sequence coding for the epitope recognized by the anti c-myc monoclonal antibody 9E10 has been inserted at the N-terminus. Each of the expressed proteins has this epitope as a tag, allowing its localization by light and electron microscopy by the same antibody. After nuclear microinjection of these plasmids into MDCK or Rat 2 cells, expression of the protein (6-18 hr later) was confirmed by immunofluorescence labeling with 9E10 imaged by confocal microscopy. For ultrastructural localization of these tagged proteins, a method was devised to process microinjected cells in situ into low-temperature resin. The proteins were localized on the sections using 9E10 detected with colloidal gold conjugates. Ha-ras protein was localized almost exclusively on the cell membranes. Rho-A and rho-C were predominantly associated with the submembraneous actin network, and rho-B was found in association with multivesicular bodies. Rac1 protein induces the formation of large pinocytotic vesicles and was detected on the cytoplasmic face of these vacuoles. These experiments demonstrate the successful use of this approach for detection of de novo synthesized proteins from microinjected plasmids by both light and electron microscopy on a small (< 50 cells) sample size.
Dopamine D1 receptor distribution in Sf9 cells imaged by confocal microscopy: a quantitative evaluation.
Authors: Trogadis J E , Ng G Y , O'Dowd B F , George S R , Stevens J K
Published: April 2011

A c-myc epitope-tagged human dopamine D1 receptor (c-myc D1 receptor) was expressed in Sf9 cells and its cellular distribution under basal conditions and after exposure to the agonist dopamine was examined. In the basal state, immunofluorescently labeled c-myc D1 receptors imaged by confocal microscopy appeared as a bright ring of label predominantly on the cell surface, and to a lesser extent as intracellular clusters of label. This pattern of receptor distribution was confirmed by radioligand-binding assays on plasma membrane and light membrane fractions using the D1 receptor-antagonist [3H]-SCH-23390. After exposure to dopamine, c-myc D1 receptors were redistributed on the cell surface, changing from a continuous ring to a discontinuous pattern of label. Analysis of fluorescence intensity and three-dimensional computer reconstruction of labeled receptors revealed a 30% decrease in surface labeling with no decrease in total number of receptors confirmed by radioligand-binding analysis. These findings constituted the first direct evidence of agonist-induced D1 receptor internalization. The results showed that the combination of confocal microscopy and three-dimensional reconstruction can be used to visualize and assess receptor distribution in Sf9 cells.
Differential expression of cathepsins B and D in testis and epididymis of adult rats.
Authors: Igdoura S A , Morales C R , Hermo L
Published: April 2011

Cathepsins are specific proteases in lysosomes that participate in the degradation of proteins, some of which are derived from endocytosis. In this study we examined the immunocytochemical localization of cathepsin B and D antibodies in cells of rat testis and epididymis, using light and electron microscopic immunocytochemistry. In testis, cathepsin D was immunolocalized over lysosomes of Sertoli cells and Leydig cells and on the acrosome of spermatids. Cathepsin B was found over lysosomes of macrophages. Non-ciliated cells of the efferent ducts revealed intense immunogold labeling over lysosomes with both anti-cathepsin B and D antibodies. In epididymis, cathepsins B and D showed marked variations in expression over the different epithelial cells and regional differences for a given cell type. Anti-cathepsin D antibodies showed intense labeling over lysosomes of principal cells in the corpus and proximal cauda. In contrast, anti-cathepsin B antibodies revealed intensely labeled lysosomes of principal cells of the distal initial segment, intermediate zone, and caput epididymidis, with weaker labeling in other regions. Clear cells of the proximal caput epididymidis revealed intensely labeled lysosomes for anti-cathepsin D antibodies. In the distal caput, clear cells showed a variable reaction pattern from intensely labeled to unreactive. Basal cells of teh intermediate zone and proximal caput region were intensely reactive for anti-cathepsin D antibodies. There was no staining over clear or basal cells with anti-cathepsin B antibodies. Taken together, these results demonstrate cell-specific and regional differences in the distribution of cathepsins B and D in cells of the male reproductive system. Such results suggest substrate specificity with regard to protein turnover within lysosomes of cells of testis and epididymis.
The gene encoding vitamin K-dependent anticoagulant protein C is expressed in human male reproductive tissues.
Authors: He X , Shen L , Bjartell A , Malm J , Lilja H , Dahlbäck B
Published: April 2011

Protein C is a vitamin K-dependent protein circulating in plasma as a zymogen to an anticoagulant serine protease. After its activation, protein C cleaves and inactivates coagulation factors Va and VIIIa. Human protein C is synthesized in liver and undergoes extensive post-translational modification during its synthesis. Recently, the protein C inhibitor was demonstrated to be synthesized in several organs of the human male reproductive tract. Moreover, vitamin K-dependent protein S, which functions as a co-factor to activated protein C, was found to be synthesized in the Leydig cells of human testis. The aim of this study was to elucidate whether the protein C gene is also expressed in the male reproductive system. Specific immunostaining of protein C was found in Leydig cells of human testis, in the excretory epithelium of epididymis, and in some epithelial glands of the prostate, whereas no immunostaining was detected in seminal vesicles. Northern blotting and non-radioactive in situ hybridization demonstrated protein C mRNA in Leydig cells, in the excretory epithelium of epididymis, and in some of the epithelial glands of the prostate. The mRNA was distributed perinuclearly and the localization was in accordance with the specific immunostaining for protein C. The epithelium of epididymis was also found to contain both protein S mRNA and immunoreactivity. The demonstration of both protein C and protein S immunoreactivities, as well as their mRNAs, in male reproductive tissues suggests as yet unknown local functions for these proteins.
Measurement of cAMP in the cardiac conduction system of rats.
Authors: Sugiyama A , McKnite S , Wiegn P , Lurie K G
Published: April 2011

To characterize differences in regional cAMP production in the cardiac conduction system, 18 rats were anesthetized with pentobarbital (65 mg/kg IP) and randomized into a control (n = 9) and a stimulated group (n = 9). The stimulated group received aminophylline (20 mg/kg SC) and isoproterenol (16 micrograms/kg SC). The concentration of cAMP in freeze-dried, micro dissected pieces (1-3 micrograms) of cardiac tissue was measured using a new microanalytical method. The cAMP contents in right atrium, atrioventricular node, His bundle, and left ventricle (fmol/microgram dry weight, mean +/- SE) were 38.9 +/- 2.5, 39.0 +/- 4.3, 46.4 +/- 6.1, and 41.4 +/- 3.3 in controls and 72.9 +/- 6.7, 86.1 +/- 2.9, 115.0 +/- 11.5, and 79.5 +/- 7.3 in the stimulated group, respectively. Basal cAMP levels were similar throughout the heart, whereas isoproterenol increased cAMP levels in all regions (p < 0.01). Furthermore, cAMP levels in His bundle, after isoproterenol, were higher than in any other region (p < 0.05). These results demonstrate that: (a) cAMP can be measured in discrete portions of the cardiac conduction system; (b) there are significant regional differences of beta-adrenergic control in the cardiac conduction system; and (c) cAMP production after beta-adrenergic stimulation was lower than expected in the AV nodal region, based on previously described beta-adrenoceptor density measurements.
Expression of human mucous proteinase inhibitor in respiratory tract: a study by in situ hybridization.
Authors: Van Seuningen I , Audie J P , Gosselin B , Lafitte J J , Davril M
Published: April 2011

Human mucous proteinase inhibitor (MPI) is present in bronchial secretions, where it participates in the protection of lung structures against degradation by leukocyte elastase. The protein has been localized by immunohistochemical studies and immunogold labeling essentially in the serous cells of the submucosal glands and also in the surface epithelial cells of central and peripheral airways. However, until now no gene expression study has been performed at the tissue level. In this study, in situ hybridization was used to precisely study MPI mRNA expression in bronchial tissue sections with a specific radiolabeled oligonucleotide probe. By light microscopy, MPI gene expression was localized exclusively in the serous and seromucinous acini of the submucosal glands of large airways. The MPI gene was expressed in submucosal glands of normal and carcinomatous tissue sections, whereas it was not expressed in bronchial and bronchiolar surface epithelia.
Fluorescence overlay antigen mapping of the epidermal basement membrane zone: III. Topographic staining and effective resolution.
Authors: Bruins S , De Jong M C , Heeres K , Wilkinson M H , Jonkman M F , Van der Meer J B
Published: April 2011

In this third study on the fluorescence overlay antigen mapping (FOAM) technique, we have addressed the question of which differences of antigen distributions close to the resolving power of the light microscope can be distinguished. An answer to this question should provide clues to future applications of the technique aiming at the topographic differentiation of IgG deposits displayed at the epidermal basement membrane zone (EBMZ) in certain bullous skin disorders. For the present purpose we have developed a topographic staining model in human skin, using structural EBMZ antigens as topographic reference markers. The distribution of these markers relative to one another is visualized in FOAM images obtained by selective double immunofluorescence tracing and videomicroscopic overlay imaging. The theoretical resolution limit of the technique is discussed and suggests an effective lower limit of some 60-65 nm. Although this limit is not reached under present conditions, our results show that it is possible to distinguish topographic differences of antigen distributions with an upper resolution limit of 200 +/- 50 nm. Furthermore, our findings indicate that collagen Type VII and beta 4 integrin are the most suitable molecules to serve as topographic reference markers in future applications of the technique aiming at the differentiation of bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). Preliminary results on this topic are most promising indeed.
Association of hyaluronan with rat vascular endothelial and smooth muscle cells.
Authors: Eggli P S , Graber W
Published: April 2011

We investigated the distribution of hyaluronan (hyaluronic acid) in rat vascular tissue fixed by an osmium tetroxide (or glutaraldehyde) microwave technique and embedded in epoxy resin (or Lowicryl K4M), using hyaluronan binding proteins coupled to 15-20-nm gold particles as ultrastructural markers in a one-step post-embedding procedure. The intra- and extracellular aspects of vascular endothelial and smooth muscle cell plasma membranes revealed distinct labeling, with a high affinity for caveolae being manifested in both cell types and in all kinds of vessels. Hyaluronan was also localized intercellularly in areas characterized by extensive endothelial cell interdigitation. Intracellularly, moderate staining of nuclear heterochromatin was observed.
Fluorescence overlay antigen mapping of the epidermal basement membrane zone: II. Color fidelity.
Authors: Bruins S , De Jong M C , Heeres K , Wilkinson M H , Jonkman M F , Van der Meer J B
Published: April 2011

In this second report on the fluorescence overlay antigen mapping (FOAM) technique, we highlight some of the errors that may influence faithful color rendition of slide preparations using triple antigen immunofluorescence staining. Reliable interpretation of multicolor fluorescence images requires that the observer can unambiguously assign each color in these images to the presence of a specific combination of the labeled antigens. This is possible only when the image fidelity meets certain standards. The present study concentrates on color fidelity which is easily undermined by spectral matching errors, image contrast errors, and exposure time errors. Evaluation of these errors, using the photomicrographic overlay variant of FOAM, showed the potential unreliability of the simultaneous use of multiple fluorophores for immunofluorescence microscopy. The procedures described here may serve as a solid starting point in formulating technical conditions that allow reliable color rendition in multicolor immunofluorescence microscopy. Furthermore, these procedures can be adapted to studies other than the analysis of basement membrane zone antigens, to which they have been first applied.
Rapid processing of filter-grown cells for Epon embedding.
Authors: Parton R G
Published: April 2011

A simple and rapid method for fixation, processing, and Epon embedding of cells grown on a filter support is described. The results show that it is possible to embed filter-grown cells in resin within 30 min of applying the primary fixative and still to achieve good ultrastructural preservation.
Non-radioactive localization of nucleic acids by direct in situ PCR and in situ RT-PCR in paraffin-embedded sections.
Authors: Martínez A , Miller M J , Quinn K , Unsworth E J , Ebina M , Cuttitta F
Published: April 2011

Technological developments have made possible extension of polymerase chain reaction (PCR) analysis to individual cells to localize DNA/RNA with non-radioactive labels at the light microscopic level. This approach, in situ PCR, is particularly useful in resolving low-frequency message expression in mixed populations of cells and tissues. We have established a working protocol for direct in situ PCR and have utilized several controls to validate our results. In this report we outline the procedures for detecting either DNA or RNA in a rapid and reproducible manner. We evaluate the sequential steps required for this analysis, such as protease hydrolysis, DNAse digestion, "hot start" capabilities, and detection methods. We have applied these methods in several applications, including detection of the p53 gene in human tumor samples, localization of insulin-like growth factor-IA mRNA in cell lines with low levels of expression, and distribution of transferrin mRNA in lung cancer cell lines and tumors. We demonstrate from this study that the in situ PCR technique is an investigative approach capable of detecting specific DNA/RNA sequences at the cellular level and of identifying cells with low levels of mRNA expression.
Intermediate filaments in cardiac myogenesis: nestin in the developing mouse heart.
Authors: Kachinsky A M , Dominov J A , Miller J B
Published: April 2011

By using immunohistology combined with immunoblotting, cell culture, and RT-PCR, we show that the intermediate filament protein nestin is transiently expressed in the midembryonic mouse heart. Monoclonal antibody (MAb) Rat-401, known to react with nestin in neural and skeletal muscle cells, was also found to react with ventricular and atrial cells throughout the mouse heart from embryonic day 9 (E9) through E10.5. Both before (E8.5) and after (E11-adult) this brief period, staining with Rat-401 was absent from atrial and ventricular myocytes. To evaluate the specificity of staining with MAb Rat-401 in the heart, we used immunoblotting, cell culture, and RT-PCR to verify that the authentic nestin protein and mRNA were expressed in cardiomyocytes of the E10 mouse. Nestin expression is the first molecular marker for this distinct midembryonic period of heart development.
Possibilities of false immunocytochemical results generated by the use of monoclonal antibodies: the example of the anti-proinsulin antibody.
Authors: Bendayan M
Published: April 2011

The immunocytochemical application of a monoclonal antibody (MAb) against the Arg-Arg region at the junction of the C-peptide and the insulin beta-chain of the human proinsulin molecule on rat pancreatic tissue resulted in positive immunogold labeling over the insulin- as well as the glucagon-secreting cells. In the insulin cells, the Golgi cisternae and the immature clathrin-coated granules were intensely labeled, and in the glucagon cells all the granules were labeled. Control experiments using the anti-proinsulin MAb adsorbed with proinsulin, insulin, and glucagon have confirmed the crossreactivity of this proinsulin antibody with glucagon. Furthermore, the anti-proinsulin MAb appears to crossreact not only with rat but also with human glucagon and with bovine and porcine insulin and glucagon. Examination of the amino acid sequences of proinsulin and proglucagon has shown that both molecules display this Arg-Arg dipeptide sequence, which could explain the labeling obtained on both cell types. In addition, other propeptides also display such a sequence and, indeed, positive cells were detected in rat pituitary. These results demonstrate that by immunocytochemistry this MAb, although providing very specific results, reveals non-related molecules. This is due to the fact that these molecules exhibit similar short determinants. MAbs raised against very short amino acid sequences can reveal this determinant in various molecules, generating false-positive results.
Effect of light stimulation on the cytochemical localization of cytochrome C oxidase in photoreceptor cells of cichlid fish.
Authors: Körtje K H , Körtje D , Rahmann H
Published: April 2011

Cytochrome oxidase activity was evaluated cytochemically in rod photoreceptor cells in the retina of the cichlid fish Oreochromis mossambicus after different stimulation protocols. The enzyme activity was assessed semiquantitatively by estimating the volume ratio of mitochondria classified according to the intensity of enzyme reactivity. Dark adaptation for 5 hr induced an increase of cytochrome oxidase activity both in vivo and in vitro, i.e., in isolated retinas. Short-term illumination (1 hr) of isolated retinas adapted previously in vivo to darkness caused a significant decrease of enzyme activity, whereas short-term darkening after in vivo light adaptation had no effect. Chemical stimulation for 15 min with increased K+ concentration (20 mM) reduced the enzyme activity, i.e., chemical depolarization did not have the same effect as depolarization induced by darkening. Significant changes in cytochrome oxidase activity were apparent within 1 hr of stimulation, so that this method for analysis of neuronal activity can be applied even in short-term experiments.
Cytotrophoblast cells: a barrier to maternofetal transmission of passive immunity.
Authors: Bright N A , Ockleford C D
Published: April 2011

The human fetus receives passive immunity via the chorioallantoic placenta in the form of maternal immunoglobulin G (IgG) class antibodies. This provides protection against pathogens at a time when the fetus is immunologically naive. We localized endogenous human IgG using confocal laser scanning fluorescence microscopy and immunoelectron microscopy of frozen sections of chorionic villi from early and late gestation. With confocal microscopy we also investigated the distribution of a receptor for IgG (Fc gamma RIII; CD16) that is typically expressed on the surface of human leukocytes. Endogenous IgG was present in the syncytiotrophoblast that surrounds chorionic villi but underlying cytotrophoblast cells were devoid of endogenous antibody. Fc gamma RIII immunoreactivity was confined to the syncytiotrophoblast and was also absent from cytotrophoblast cells. We propose that cytotrophoblast cells present a barrier to the transmission of maternally derived IgG across the human placenta. This accounts for the paradox that there are low levels of transport in the first trimester when the syncytiotrophoblast is known to express receptors for IgG. Cytotrophoblast cells form an almost complete epithelial layer underlying the syncytiotrophoblast at this stage of gestation, but this becomes discontinuous as the placenta matures, thus removing the cellular impediment to IgG transmission.
Differential subcellular localization of neural isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin) in central nervous system neurons: immunohistochemistry on formalin-fixed paraffin sections employing antigen retrieval by microwave irradiation.
Authors: Usuda N , Arai H , Sasaki H , Hanai T , Nagata T , Muramatsu T , Kincaid R L , Higuchi S
Published: April 2011

We examined the immunohistochemical distribution of the two mammalian isoforms of calcineurin catalyic subunits, A alpha and A beta, in central nervous system (CNS) tissues of cows, rats, and humans. Cryostat sections and paraffin sections of parformaldehyde-fixed tissues were stained with antipeptide antibodies for each isoform. The same localization pattern was observed in both cryostat and paraffin sections. In the latter, the intensity of the staining was dramatically enhanced by microwave irradiation. Calcineurin isoforms were localized in a variety of nerve cells but not in neuroglial cells. Their differential expression as the A alpha isoform in the nucleus and the A beta isoform in the cytoplasm was present in a variety of CNS nerve cells, most distinctively in Purkinje cells of the cerebellum and pyramidal cells of the cerebrum, irrespective of species. These results suggest that each isform has distinct intracellular sites of action in CNS neurons and that the phenomenon has been conserved during mammalian evolution.
Distribution of peptidyl-glycine alpha-amidating mono-oxygenase (PAM) enzymes in normal human lung and in lung epithelial tumors.
Authors: Saldise L , Martínez A , Montuenga L M , Treston A , Springall D R , Polak J M , Vázquez J J
Published: April 2011

C-terminal alpha-amidation is a post-translational modification necessary for the biological activity of many regulatory peptides produced in the respiratory tract. This modification is a two-step process catalyzed by two separate enzyme activities, both derived from the peptidyl-glycine alpha-amidating mono-oxygenase (PAM) precursor. The distribution of these two enzymes, peptidyl-glycine alpha-hydroxylating monoxygenase (PHM) and peptidyl-alpha-hydroxyglycine a amidating lyase (PAL), was studied in the normal lung and in lung tumors using immunocytochemical methods and in situ hybridization. In normal lung the enzymes were located in some cells of the airway epithelium and glands, the endothelium of blood vessels, some chondrocytes of the bronchial cartilage, the alveolar macrophages, smooth muscle cells, neurons of the intrinsic ganglia, and in myelinated nerves. A total of 24 lung tumors of seven different histological types were studied. All cases contained PAM-immunoreactive cells with various patterns of distribution. All immunoreactive cells were positive for the PHM antiserum but only some of them for the PAL antiserum. The distribution of PAM co-localizes with some other previously described amidated peptides, suggesting that amidation is an important physiological process taking place in the normal and malignant human lung tissue.
CD66a (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues.
Authors: Prall F , Nollau P , Neumaier M , Haubeck H D , Drzeniek Z , Helmchen U , Löning T , Wagener C
Published: April 2011

CD66a, also called biliary glycoprotein (BGP), is a member of the carcinoembryonic antigen (CEA) family and of the immunoglobulin superfamily. CD66a is the human homologue of Cell-CAM, a well-defined cell adhesion molecule of the rat. In the present study a monoclonal antibody specific for CD66a was used to locate CD66a in human tissues. CD66a is expressed in epithelia, in certain endothelia, and in cells of the myeloid lineage. Hepatocytes were stained along the bile canaliculi. A characteristic apical membranous staining was observed in enterocytes, superficial absorptive cells of the colon, in the epithelia of esophageal and Brunner's glands, bile ducts and gallbladder, pancreatic ducts, proximal tubules of the kidney, prostate, endometrium, and mammary ducts. Selective staining of endothelia was present in glomeruli and vasa recta of the kidney, small placental vessels, adrenal sinusoids, endometrium, the prostate. Among the cells of the myeloid lineage, granulocytes and myelocytes were positive. The expression of CD66a by human cells and tissues is well comparable with the expression reported for Cell-CAM, the rat counterpart of CD66a. The wide tissue distribution of CD66a indicates that CD66a is a prominent human adhesion molecule.
Differential expression of gap junction proteins connexin32 and 43 in rat submandibular and sublingual glands.
Authors: Muramatsu T , Hashimoto S , Shimono M
Published: April 2011

We examined the expression and localization of the gap junction proteins connexin32 and 43 in rat submandibular and sublingual glands. Western blot analysis with anti-connexin32 and 43 antibodies showed bands of approximately 27 KD and 43 KD, respectively, in both glands. Immunofluorescence microscopy demonstrated the presence of reactive spots for connexin32 between acinar cells in both glands. The frequency of connexin32-positive spots in the submandibular glands was approximately equal to that in the sublingual glands. In contrast, reactive spots for connexin43 were observed at the periphery of the alveolar structures in both glands. The connexin43-positive spots in the sublingual glands were more frequent and larger than those in the submandibular glands. No positive spots for both connexins were detected between duct cells in either gland. Immunoelectron microscopy revealed that connexin32 was localized to the gap junctional membranes between acinar cells. Immunolabeling for connexin43 was located on the gap junctions between thin processes of myoepithelial cells. These results suggest that connexin32 of the gap junction is associated with regulation of the secretory function of acinar cells and that connexin43 is associated with that of contraction of the myoepithelial cells in rat salivary glands.
Ultrastructural immunogold localization of interleukin 5 to the crystalloid core compartment of eosinophil secondary granules in patients with atopic asthma.
Authors: Möller G M , de Jong T A , Overbeek S E , van der Kwast T H , Postma D S , Hoogsteden H C
Published: April 2011

Bronchial biopsies from two patients with atopic asthma were analyzed by immunogold labeling to detect the ultrastructural location of interleukin 5 (IL-5). In eosinophils, IL-5 was localized to the electron-dense crystalloid core compartment of the secondary or specific eosinophil granules. Other structures in the eosinophils were unlabeled. No IL-5 was detected in mast cells. Control sections incubated with an irrelevant primary antibody were negative. This study demonstrates that pre-formed IL-5 is stored within the major population of secondary granules in human eosinophils.
Preservation of spatial organization and antigenicity of leukocyte surface molecules by aldehyde fixation: flow cytometry and high-resolution FESEM studies of CD62L, CD11b, and Thy-1.
Authors: Hasslen S R , Burns A R , Simon S I , Smith C W , Starr K , Barclay A N , Michie S A , Nelson R D , Erlandsen S L
Published: April 2011

We used transmission and scanning electron microscopy in conjunction with immunogold labeling to study cell surface molecules for evidence of distribution-function relationships. Ascription of functional significance to surface distribution therefore requires preservation of cell morphology and maintenance of molecular expression and distribution through the multiple steps of cell preparation. These requirements prompted us to compare two methods for preparing leukocytes for analysis of surface molecule distribution: one method involved using low temperature to "stabilize" cell morphology and surface molecular organization through immunolabeling; the other involved fixation of the cells with dilute glutaraldehyde before their isolation and labeling. Binding of primary antibodies to several surface molecules, measured by flow cytometry, was comparable for cells prepared by the two methods. Cell morphology and molecular distributions, assessed by high-resolution field emission SEM, were likewise comparable. These results support the conclusion that cell morphologies and CAM distributions previously reported were not affected by exposure of the cells to low temperature through isolation and immunolabeling. Our additional observation that Thy-1 is expressed on both non-projecting and projecting membrane domains of mouse lymph node lymphocytes and rat thymocytes represents a third and new pattern of surface molecule distribution.
NeuN: a useful neuronal marker for diagnostic histopathology.
Authors: Wolf H K , Buslei R , Schmidt-Kastner R , Schmidt-Kastner P K , Pietsch T , Wiestler O D , Blümcke I
Published: April 2011

The monoclonal antibody A60 specifically recognizes the DNA-binding, neuron-specific protein NeuN, which is present in most neuronal cell types of vertebrates. In this study we demonstrate the potential use of NeuN as a diagnostic neuronal marker using a wide range of formalin-fixed, paraffin-embedded human surgical and autopsy specimens from the central and peripheral nervous system. After microwave antigen retrieval, almost all neuronal populations revealed strong immunoreactivity for NeuN in nuclei, perikarya, and some proximal neuronal processes, whereas more distal axon cylinders and dendritic ramifications were not stained. The stain greatly enhanced the gray matter architecture. NeuN immunoreactivity was not detected in Purkinje cells, most neurons of the internal nuclear layer of the retina, and in sympathetic chain ganglia. We examined nine gangliogliomas and 14 dysembryoplastic neuroepithelial tumors, one ganglioneuroma, and one dysplastic cerebellar gangliocytoma. The neuronal component of all of these lesions showed marked immunoreactivity for NeuN. In addition, NeuN immunoreactivity was focally seen in one of seven medulloblastomas with prominent neuronal differentiation. There was no staining of non-neuronal structures. The results indicate that NeuN immunoreactivity is a sensitive and specific neuronal marker in formalin-fixed, paraffin-embedded tissues, and may be useful in diagnostic histopathology.
Functional localization of glucose transporter 2 in rat liver.
Authors: Ogawa A , Kurita K , Ikezawa Y , Igarashi M , Kuzumaki T , Daimon M , Kato T , Yamatani K , Sasaki H
Published: April 2011

Heterogeneity of zonal hepatocytes is important to elicit specific liver function. We investigated the distribution of glucose transporter 2 (GLUT-2) in normal rat liver by immunostaining and Northern blot analysis. GLUT-2 stained by immunohistochemistry was distributed predominantly in the periportal hepatocytes and gradually thinned towards the perivenous zone. Ultrastructural immunostaining of GLUT-2 showed that it was localized on microvilli of the sinusoidal plasma membrane of hepatocytes but not on the basolateral plasma membrane. Consistent with the distribution of GLUT-2 protein, the level of GLUT-2 mRNA in periportal hepatocytes was 1.9-fold higher than in perivenous hepatocytes selectively isolated by the differential isolation technique. In addition, the mRNA level of phosphoenolpyruvate carboxykinase, one of the key enzymes of gluconeogenesis, was also twofold higher in the periportal hepatocytes. These results suggest that GLUT-2 contributes to the functional difference between periportal and perivenous hepatocytes in glucose metabolism of the liver.
Quantitative radioimmunohistochemical measurements of p185(erbB-2) in frozen tissue sections.
Authors: Reeves J R , Going J J , Smith G , Cooke T G , Ozanne B W , Stanton P D
Published: April 2011

The relationship between expression of the c-erbB-2 proto-oncogene and the biology of breast cancer has been investigated widely, most studies using immunohistochemistry in formalin-fixed, paraffin-embedded tissues. This technique is at best semiquantitative and there is a high degree of interstudy variability because of its subjective nature and poor methodological standardization. The relationship between the levels of expression and biology can be examined thoroughly only with an accurately quantitative technique. We have developed a radioimmunohistochemical assay to measure p185(erbB-2) in tissue biopsy specimens. The method involves incubating frozen sections with 125I-labeled monoclonal antibody, microautoradiograpy, and grain counting with image analysis. Sections of cell pellets with known c-erbB-2 levels are processed with each batch of samples as internal calibration standards. We have quantified c-erbB-2 expression in 60 breast carcinomas and compared the results with conventional immunohistochemistry. Radioimmunohistochemistry measured receptor levels throughout the range of expression in breast carcinomas, whereas conventional immunohistochemistry detected the protein only in the highest expressing tumors. The quantitative, objective data produced by radioimmunohistochemistry allow a more thorough evaluation of the relationship between c-erbB-2 expression and tumor biology. This technique may have applications in other fields where quantitative data is required and relevant monoclonal antibodies are available.
Classical morphology, esterase cytochemistry, and interphase cytogenetics of peripheral blood and bone marrow smears.
Authors: Jacobsson B , Bernell P , Arvidsson I , Hast R
Published: April 2011

We used peripheral blood (PB) and bone marrow (BM) smears in the development of two methods based on cytomorphology and esterase cytochemistry in combination with fluorescence in situ hybridization (FISH). The first method involves photodocumentation of May-Grünewald-Giemsa (MGG)-stained cells, followed by destaining in methanol-acetic acid, fixation in paraformaldehyde, and digestion with protease and RNAse before FISH using alpha-satellite probes that specify chromosomes X, 7, 8, and 17. On average, two hybridization signals were seen in 94.5% of disomic BM cells. The hybridization sensitivity was found to vary, however, both among morphologically defined hematopoietic cell lineages and among differentation levels within a lineage. In the second method, an esterase staining technique was followed by the same treatment as for MGG-stained cells. The esterases and FISH signals could be simultaneously visualized and the method was found suitable for rapid screening of in situ signals in cytochemically defined granulocytes and lymphocytes but not in monocytes. The combined methods proved very useful in elucidating the clinical significance of chromosomal abnormalities seen in two cases of leukemia.
mRNA expressions of TIMP-1, -2, and -3 and 92-KD type IV collagenase in early human placenta and decidual membrane as studied by in situ hybridization.
Authors: Hurskainen T , Höyhtyä M , Tuuttila A , Oikarinen A , Autio-Harmainen H
Published: April 2011

Cytotrophoblasts of early placenta invade the decidual membrane, gestational endometrium, and spiral arteries during early pregnancy. Unlike tumor invasion, this physiological invasion is well controlled, although its molecular mechanisms are largely unknown. We have previously shown that cytotrophoblasts synthesize significant mRNAs for 72-KD Type IV collagenase, laminin, and Type IV collagen, proteins implicated in extracellular matrix turnover and migration. In this study we used in situ hybridization and immunohistochemistry to investigate the mRNA expression pattern of 92-KD Type IV collagenase and the matix metalloproteinase inhibitors TIMP-1, TIMP-2, and TIMP-3 in early human placenta and decidual membrane. mRNAs for 92-KD Type IV collagenase, TIMP-1, TIMP-2, and TIMP-3 were found in the cells of cytotrophoblastic columns, the endothelial and fibroblastic stromal cells of villi, and the large decidualized cells of decidual membrane. TIMP-1 expression was notably accentuated in the fibroblasts of fibrotic villi. In the decidual membrane, the signals for 92-KD Type IV collagenase and TIMP-1 mRNA were particularly strong around the glandular structures. The trophoblastic epithelium of villi and the epithelial cells of decidual glands showed a signal for 92-KD Type IV collagenase and TIMP-2, but not for TIMP-1 or TIMP-3. The coincidental expression of the proteolytic 92-KD Type IV collagenase and inhibitors TIMP-1, TIMP-2, and TIMP-3 generally in the same cells suggests that the activity of 92-KD Type IV collagenase, which is regulated by TIMPs, plays an important role in placental tissue organization and in the invasion of trophoblastic cells into the uterine wall.
Expression of retinoic acid receptor proteins in basal cell carcinomas: an immunohistochemical analysis.
Authors: Kamradt J , Reichrath J
Published: April 2011

We analyzed immunohistochemically the expression of RAR proteins in basal cell carcinomas (BCCs; n = 15) in situ. The labeling pattern for the different types of RARs was compared with the staining pattern of the proliferation marker Ki-67 in the same tumors. We found strong immunoreactivity for RAR-alpha and moderate immunoreactivity for RAR-gamma in all BCCs analyzed, whereas no or very weak staining for RAR-beta protein was detected. In contrast to RAR-gamma, which revealed no or only marginal differences in staining intensities, RAR-alpha immunoreactivity was consistently stronger in BCCs compared to adjacent unaffected epidermis. In general, labeling of BCCs for RAR-alpha and RAR-gamma was pronounced in cells of the palisade and peripheral cells, whereas staining in the center of the tumors was heterogeneous. Eleven of the 15 BCCs analyzed revealed no visual correlation in comparing labeling patterns for RAR-alpha and RAR-gamma with the labeling pattern for Ki-67. In four specimens, expression of RAR-alpha, RAR-gamma, and Ki-67 proteins was confined to peripheral tumor cells. Our findings indicate that (a) RAR-alpha and RAR-gamma proteins are, in contrast to RAR-beta, strongly expressed in BCCs; (b) expression of RAR-alpha is upregulated in BCCs compared to keratinocytes of uninvolved epidermis; and (c) BCCs may be targets for potentially preventive or therapeutic treatment with RAR-alpha- or RAR-gamma-selective retinoic acid metabolites.
Ontogeny of Clara cell-specific protein and its mRNA: their association with neuroepithelial bodies in human fetal lung and in bronchopulmonary dysplasia.
Authors: Khoor A , Gray M E , Singh G , Stahlman M T
Published: April 2011

Clara cell-specific 10-KD protein (CCSP) is an abundant product of nonciliated bronchiolar epithelial (Clara) cells in the lung. We have determined the temporal-spatial distribution of CCSP and its mRNA in developing human lung and in neonatal lung disease, using immunohistochemistry and in situ hybridization. CCSP immunoreactivity was found in nonciliated bronchiolar epithelial cells from 12 weeks of gestation onward. Tracheal and bronchial epithelia showed positive immunoreactivity at each gestational week after 15 weeks and 14 weeks, respectively. CCSP mRNA was seen in the bronchial and bronchiolar epithelia from 16 weeks onward and was detected in the trachea from 19 through 23 weeks of gestation. CCSP immunoreactivity and mRNA were present in nonciliated single cells of bronchial and bronchiolar epithelia in fetuses and in infants with and without lung disease. CCSP- and CCSP mRNA-containing epithelial cells also formed dusters around neuroepithelial bodies (NEBs), especially at airway branch points, suggesting that NEBs and Clara cells might interact during development and during pulmonary regeneration. Because of evidence of overlapping of some but not all cells expressing CCSP, SP-A, and pro-SP-B during lung development, a common cell lineage is proposed, with subsequent divergence of phenotypes.
Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures.
Authors: Tanner V A , Ploug T , Tao-Cheng J H
Published: April 2011

We demonstrated the subcellular localization of SV2, a transmembrane protein associated with neuroendocrine secretory vesicles, in NGF-treated PC12 cells by preembedding EM immunocytochemistry (ICC), using a small gold probe followed by silver enhancement. The use of a multiwell chamber slide substantially improved the efficiency of the preembedding EM ICC procedures for cell cultures. The advantages and related caveats of this method are discussed. SV2 was distinctly localized on dusters of synaptic vesicles and large dense-cored vesicles (LDCV). The distribution of SV2 on these two types of secretory vesicles was compared quantitatively to that of another secretory vesicle-associated transmembrane protein, synaptophysin. In cultures under similar experimental conditions, the ratio of SV2 vs synaptophysin ICC staining on synaptic vesicle dusters was about 1:1, whereas it was about 9:1 on LDCV membranes. Furthermore, whereas SV2 is localized on the membranes of the LDCVs, chromogranin A, an acidic protein in secretory granules, is clearly in the core of the LDCVs. This is the first demonstration of these two antigens in such dose (approximately 20 nm) yet distinct compartments within a single organelle.
Enhancement of TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method using mung bean nuclease, a single-stranded DNA digestion enzyme.
Authors: Umemura S , Yasuda M , Osamura R Y , Kawarada Y , Sugiyama T , Tsutsumi Y
Published: April 2011

The TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method has been employed widely to demonstrate apoptotic cells in routinely prepared paraffin sections. Because the apoptotic cells were reactive with the antibody to single-stranded DNA, we attempted to enhance the TUNEL positivity by pretreatment with single-stranded DNA digestion enzymes, S1 nuclease, and mung bean nuclease. When mung bean nuclease (5 U/50 microliter/section) was incubated at 37 degrees C for 30 min, the TUNEL reaction was most effectively enhanced. Pretreatment with S1 nuclease (0.25 U/50 microliter/section) at 37 degrees C for 45 min was less reliable. Compared with the conventional TUNEL sequence, the enhancement technique using mung bean nuclease enabled us to detect more apoptotic cells in human small intestine, colon, tonsil, thymus, endometrium, ovary, liver, kidney, and pancreas. The positivity was not affected by autolytic change. The mechanism of enhancement is discussed.