bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine.
Authors: Kerstens H M , Poddighe P J , Hanselaar A G
Published: April 2011

For amplification of in situ hybridization (ISH) signals, we describe a method using catalyzed reporter deposition (CARD). This amplification method is based on the deposition of biotinylated tyramine (BT) at the location of the DNA probe. The BT precipitate can then visualized with fluorochrome- or enzyme-labeled avidin. Both for bright-field ISH (BRISH) and for fluorescence ISH (FISH), the detection limit was highly increased. This method is especially suitable for visualization of very weak ISH signals, such as those obtained by ISH using locus-specific DNA probes. Furthermore, CARD amplification of ISH signals (CARD-ISH) is highly sensitive, rapid, flexible, and easy to implement. Successful application of CARD-ISH with locus-specific DNA probes on histological and cytological samples may improve the determination of structural chromosomal aberrations in archival material.
Morphological characterization of stromal cell types in hematopoietically active long-term murine bone marrow cultures.
Authors: Hauser S P , Waldron J A , Upuda K B , Lipschitz D A
Published: April 2011

Accurate histological evaluation of stromal morphology is very difficult in cultures incubated in plastic flasks. Employing glass flasketts, we were able to characterize the morphology and immunocytochemistry of four marrow stromal cell types in a functionally intact microenvironment of murine long-term bone marrow cultures (LTBMCs). Fibroblastoid cells stained positively for collagen Type I and III, negatively for von Willebrand factor (vWf), the mouse macrophage F4/80 antigen, and the Bandeiraea simplicifolia lectin I isolectin B4 (BSL I-B4). Endothelial cells stained positively for vWf antigen and lectin BSL I-B4 but negatively for collagen Types I and III and for F4/80 antigen. Fat-containing cells had a dense, ovaloid, indented nucleus and fat-containing vacuoles. Macrophages were strongly positive for the F4/80 antigen and stained weakly with BSL I-B4. Between the fourth and ninth weeks after culture initiation, fibroblastoid and endothelial cells remained constant, between 21 +/- 2% and 24 +/- 2% and between 3 +/- 0.3% and 4 +/- 0.4%, respectively, of the total stromal cell population. By contrast, the percentage of fat-containing cells decreased significantly from 26 +/- 3% at Week 4 to 17 +/- 2% at Week 9, and macrophages increased significantly from 49 +/- 1% at Week 4 to 57 +/- 1% at Week 9. This characterization of the stromal cell types in functionally intact LTBMCs should assist in the study of the complex interactions among the marrow stroma, cytokine production, and hematopoiesis.
In situ hybridization analyses of Na, K-ATPase alpha-subunit expression during early larval development of Artemia franciscana.
Authors: Escalante R , García-Sáez A , Sastre L
Published: April 2011

The spatial pattern of expression of the mRNA encoded by the Na,K-ATPase alpha-subunit cDNA clone pArATNa136 was determined by in situ hybridization of first, second, and third instar Artemia franciscana larvae. This mRNA was expressed at high levels in the salt gland, the antennal gland, and the end of the midgut, which are the three main osmoregulatory organs in Artemia at these stages of development. The pattern of expression was similar at the three stages of development analyzed, although the level of expression increased during development, especially in the salt and antennal glands. The expression of the mRNA coding for another Na, K-ATPase alpha-subunit isoform, the proposed alpha 2-isoform, was also determined and was shown to be limited to the salt gland. These results suggest that the clone pArATNa136 codes for the biochemically defined alpha 1-isoform of the Na,K-ATPase alpha-subunit and reinforce the importance of this isoform in osmoregulation at the three larval stages studied. The alpha 2-isoform may also be involved in osmoregulation during the first stages of larval development.
High-resolution FEISEM detection of DNA centromeric probes in HeLa metaphase chromosomes.
Authors: Rizzi E , Falconi M , Rizzoli R , Baratta B , Manzoli L , Galanzi A , Lattanzi G , Mazzotti G
Published: April 2011

HeLa metaphase chromosome spreads were hybridized with centromeric biotinylated DNA probes and detected with gold-conjugated anti-biotin antibodies. Chromosomes were observed by an in-lens field emission scanning electron microscope (FEISEM), which permits detection of biological samples without any coating. DNA probes were well localized in the centromeric region of chromosomes and there was clear discrimination between 10 nm fibers that hybridized to DNA probes and those that did not hybridize. This approach shows that in situ hybridization can be directly visualized at the FEISEM level by evaluating only secondary electron emission, which allows physical localization of the hybridized probe with high resolution so that backscatter detection represents only a control. Because chromosomes maintain the 10-nm fiber organization after in situ hybridization procedures, our data suggest that this fiber represents the lowest order of chromatin arrangement that permits transitory DNA denaturation.
Dramatic heterogeneity of transgene expression in the mammary gland of lactating mice: a model system to study the synthetic activity of mammary epithelial cells.
Authors: Faerman A , Barash I , Puzis R , Nathan M , Hurwitz D R , Shani M
Published: April 2011

We studied the expression of human serum albumin (HSA) driven by the ovine beta-lactoglobulin promoter in the mammary glands of lactating mice from five independent transgenic strains, by employing combined in situ hybridization and immunostaining techniques. Four strains displayed a heterogeneous pattern of expression: mice of strains 91 and 92 expressed the transgene in only a fraction of the lobules, whereas in strains 69 and 83 all lobules contained cells expressing HSA. In all four strains the patterns of expression within expressing lobules corresponded to the morphology of alveolar cells and to the extent of local milk secretion, suggesting that filling of alveolus with secreted material was accompanied by asynchronous downregulation of transgene expression. In situ hybridization to the endogenous milk protein genes alpha-lactalbumin, beta-casein, and whey acidic protein revealed a uniform pattern of expression in lactating mammary glands of transgeneic and in four out of five non-transgeneic mice. In the fifth control mouse, we detected downregulation of gene expression in lobules containing alveoli distended by secreted milk. The pattern of expression of the three endogenous genes was greatly disturbed after a short (3-hr) unilateral closure of mammary glands, and very much resembled the pattern of expression of the HSA transgenes. These results demonstrate that transgeneic mice provide a useful model to study the factors that regulate the synthetic activity of mammary epithelial cells.
Quantification of ANP mRNA in primary cultures of adult rat atrial myocytes by image processing: in situ hybridization to multiple parallel samples using single-stranded cDNA probes.
Authors: Kordylewski L , Ambler S K , Doyle D D
Published: April 2011

In primary cultures of adult rat atrial myocytes, we quantified the accumulation of atrial natriuretic peptide (ANP) mRNA in parallel with ANP secretion. ANP mRNA was quantified by image analysis of myocytes hybridized in situ with single-stranded cDNA probes generated by two successive thermal cycling procedures. In situ analysis permitted measurement of many small experimental samples in tandem while avoiding the possibility of differential extraction and processing of mRNA from sample to sample. The single-step application of 32P-labeled probes allowed processing of many parallel samples and generated intense punctate autoradiographic signals that were readily countable by image processing. Biotin-labeled probes, in conjunction with gold-labeled anti-biotin antibodies and silver intensification, gave an apparently equivalent specific signal but presented more difficulty in uniform processing of many samples and was harder to quantify by our image processing system. Measurement of ANP mRNA during atrial myocyte culture showed that ANP mRNA accumulated from undetectable levels after 1 day of culture to maximal levels by Day 8. In contrast, secretion of ANP (which is stored in atrial granules) slowly decreased, but was not abolished, during the first 5 days of culture. Subsequently, ANP secretion increased, with the increase trailing ANP mRNA accumulation by at least 24 hr.
Co-injection of wheat germ agglutinin-HRP and choleragenoid-HRP into the sciatic nerve of the rat blocks transganglionic transport.
Authors: Liu H , Llewellyn-Smith I J , Basbaum A I
Published: April 2011

We report on the surprising loss of transganglionic and retrograde labeling in the spinal cord of the rat after co-injection of the tracers wheat germ agglutinin-HRP (WGA-HRP) and choleragenoid toxin-HRP (CTB-HRP) into the sciatic nerve. Injection of WGA-HRP alone produced a pattern of transganglionic label consistent with transport by small-diameter primary afferent fibers. Small cell bodies were labeled in the ipsilateral dorsal root ganglion (DRG) and there was dense terminal labeling in the superficial dorsal horn of the lumbar spinal cord. Injection of CTB-HRP alone produced a pattern of transganglionic labeling consistent with transport by large-diameter primary afferent fibers. Large cell bodies were labeled in the DRG and there was dense terminal labeling in the nucleus proprius (Laminae III-V) in the spinal cord. CTB-HRP also produced extensive retrograde labeling of ventral horn motor neurons. When the two tracers were co-injected, we found few labeled cells in the ipsilateral DRG and there was almost complete loss of transganglionic terminal labeling in the lumbar spinal cord. Retrograde labeling of motor neurons was also significantly reduced. Even when one of the tracers (e.g., WGA-HRP) was injected 24 hr after and up to 10 mm proximal to the site of the first tracer (e.g., CTB-HRP), an inhibitory interaction was detected. The labeling pattern was always characteristic of the first tracer injected.(ABSTRACT TRUNCATED AT 250 WORDS)
Rapid primary microwave-aldehyde and microwave-osmium fixation: improved detection of rat parotid acinar cell secretory granule alpha-amylase using a post-embedding immunogold ultrastructural morphometric analysis.
Authors: Login G R , Ku T C , Dvorak A M
Published: April 2011

Studies of methods for improved fixation are becoming increasingly important in the field of quantitative immunocytochemistry. We used microwave (MW)-assisted chemical fixation to show improved retention of salivary gland acinar cell secretory granule alpha-amylase detected by a quantitative immunogold method. Blocks (4-mm3) of rat parotid gland were fixed by the following methods: (a) MW irradiation in an aldehyde fixative (AF) for 6 sec; (b) immersion in AF for 1.5 hr; (c) MW irradiation in osmium tetroxide (OT) for 9 sec; (d) immersion in OT for 1.5 hr; or (e) Sequential MW AF, 10 sec, MW OT rapid treatment (SMAORT), 10 sec. Specimens were processed routinely for transmission electron microscopy. Thin sections of Epon-embedded tissues were exposed first to rabbit IgG anti-human salivary alpha-amylase and second to gold-conjugated goat anti-rabbit IgG. Granule area was obtained by a point counting method. Labeling density was calculated as the number of gold particles/micron 2 +/- SD. Specimens fixed in seconds by MW-AF, MW-OT, or SMAORT showed ultrastructural preservation similar to immersion fixation in AF or OT for 1.5 hr. Immunogold labeling density of granule alpha-amylase was highest for SMAORT (874 microns 2) compared to MW-AF (295 microns 2), MW-OT (248 microns 2), routine sequential immersion in AF and OT (229 microns 2), or immersion in OT (no aldehyde) (190 microns 2). This study establishes the improved retention of salivary gland acinar cell secretory granule alpha-amylase and markedly enhanced fixation speed for ultrastructural studies made possible by MW-chemical fixation protocols that use aldehydes and osmium.
The septoclast, a cathepsin B-rich cell involved in the resorption of growth plate cartilage.
Authors: Lee E R , Lamplugh L , Shepard N L , Mort J S
Published: April 2011

At the transition between growth plate cartilage and the endochondral bone region, transverse septa are being eroded to allow the advance of invasive capillaries. To find out whether resorption is due to proteinase activity, tissue sections prepared from the growth plate/metaphyseal interface of young rats were immunostained with antibodies to the cysteine proteinase cathepsin B. Intense staining was found in a cell that is associated with the growing portion of the invasive capillaries and extends between them and the transverse septum. This cell has a single nucleus, actively synthesizes protein, and shows two other characteristic features: the cytoplasm is packed with multivesicular and dense bodies rich in cathepsin B, and the cell apex ends in a ruffled border extending into the transverse septum and signs of dissociated extracellular matrix. Even though the ruffled border resembles that of the osteoclast, the cell was not immunostained by a monoclonal antibody that recognizes a 97 KD protein known as ED1 which characterizes rat osteoclasts, monocytes, and macrophages. Therefore, this distinctive cell produces the proteinase cathepsin B and appears to be involved in the resorption of the transverse septum. The cell has been named the "septoclast."
Differentiation of human trophoblast populations involves alterations in cytokeratin patterns.
Authors: Mühlhauser J , Crescimanno C , Kasper M , Zaccheo D , Castellucci M
Published: April 2011

Cytokeratins (CKs) are related to proliferation and differentiation of epithelial cells. Little knowledge exists about CK patterns in human trophoblast subpopulations (villous and extravillous trophoblasts). To better understand differentiation and function of trophoblast components, we studied the distribution patterns of CKs in the placenta throughout pregnancy. A panel of well-defined monoclonal antibodies against different types of cytokeratins, vimentin, and fibrin, was used on frozen and paraffin sections. CK8, 18, and 19 were expressed in all the villous and extravillous trophoblastic subsets throughout pregnancy. In the first trimester, syncytiotrophoblasts were positive for CK7 and 13 along the basal membrane. As pregnancy progressed there was an increase in intensity of the reaction product and a more diffuse positive staining of CK7 in the cytoplasm of the syncytium, with evident positivity along the apical membrane. CK13 showed similar expression as CK7, but with less intense staining along the apical membrane and less prominent staining in the cytoplasm. Villous cytotrophoblasts were also positive for CK7 and CK13. CK17 was found related to cytotrophoblastic cells in contact with or next to fibrin deposits. Extravillous cytotrophoblasts in cell islands and cell columns were positive for CK13 only in the cell layers located proximal to the villous stroma, whereas the distal and more differentiated cells were negative. CK7 was positive in all epithelial cells of cell islands and columns, but the reaction product was not present in cells deeply migrated into the decidua. Amnion was negative for anti-CK13 antibodies in the first trimester but was positive at term. CK4 and CK16 were not found in the placenta. Our study shows for the first time that the different populations of human placental trophoblast express cytokeratins in developmental, differentiative, and functional specific patterns. These findings can be useful to distinguish and classify the various trophoblastic populations and provide a foundation for studying pathological aspects of the trophoblast.
Immunohistochemical localization of cytochrome P450 aromatase in equine gonads.
Authors: Almadhidi J , Seralini G E , Fresnel J , Silberzahn P , Gaillard J L
Published: April 2011

Estrogens are the major steroids produced by equine gonads. To identify the cells responsible for estrogen synthesis, an antiserum against purified equine testicular cytochrome P450 aromatase was produced in rabbits. The reactivity and specificity of the antiserum were assessed by ELISA, immunoblot analysis, and immunoneutralization studies. Immunofluorescence microscopy demonstrated that in the male gonad, cytochrome P450 aromatase (P450arom) was localized in the interstitial tissue, whereas, under the experimental conditions used, the Sertoli and germ cells did not show any specific staining. In the ovary, the granulosa cells of small follicles exhibited faint immunofluorescent staining for P450arom and the granulosa cells of large, viable more follicles showed a high degree of immunoreactivity. In the corpus luteum, all the luteinized cells showed immunoreactivity. No immunoreactivity was detected in other cells of small and large viable follicles. Immunolocalization of P450arom in the equine testicular Leydig cells and in ovarian granulosa and luteinized cells indicates that these cells have the ability to metabolize androgens to estrogens and possibly to catechol estrogens.
Localization of microsomal epoxide hydrolase in normal and neoplastic human kidney.
Authors: McKay J A , Weaver R J , Murray G I , Ewen S W , Melvin W T , Burke M D
Published: April 2011

Microsomal epoxide hydrolase is a xenobiotic metabolizing enzyme that catalyzes the conversion of toxic and carcinogenic epoxides to less toxic dihydrodiols. The cellular localization and distribution of microsomal epoxide hydrolase were investigated for the first time in normal and neoplastic human kidney. Light microscopic immunohistochemical studies using an alkaline phosphatase-anti-alkaline phosphatase technique showed that in normal kidney there was a wide distribution of epoxide hydrolase immunoreactivity. The main localization of epoxide hydrolase immunoreactivity was to the proximal and distal tubule epithelial cells. Strong epoxide hydrolase immunoreactivity was also identified in epithelium of the collecting ducts. In addition, epoxide hydrolase immunoreactivity was present in vascular endothelial cells, including endothelial cells lining glomerular capillaries. Epoxide hydrolase immunoreactivity was identified in all the renal tumors, and in each tumor immunoreactivity for epoxide hydrolase was localized to tumor cells. Immunoblotting of both normal kidney and tumor microsomes confirmed the presence of a single protein band of molecular weight 49 KD corresponding to the molecular weight of human hepatic microsomal epoxide hydrolase.
A simplified method for obtaining 0.5-microns sections of small tissue specimens embedded in PEG.
Authors: Holtham K A , Slepecky N B
Published: April 2011

We describe a new method for embedding small specimens in polyethylene glycol (PEG) 4000. This method preserves cell morphology and provides sensitive immunocytochemical labeling with excellent subcellular resolution. Small tissues are embedded in agarose so that they can be grouped together and oriented for sectioning before infiltration with PEG 4000, a water-soluble polymer. Fixation, embedding, sectioning, and staining can be performed in 1 day. Results from immunocytochemical studies localizing actin and tubulin on 0.5-micron sections of PEG-embedded specimens are compared with those obtained on semi-thin sections of araldite-embedded specimens and demonstrate the ease, speed, and increased sensitivity of this embedding method.
Monitoring morphology and signal during non-radioactive in situ hybridization procedures by reflection-contrast microscopy and transmission electron microscopy.
Authors: Macville M V , Van Dorp A G , Wiesmeijer K C , Dirks R W , Fransen J A , Raap A K
Published: April 2011

We analyzed the effects of steps in RNA in situ hybridization (ISH) procedures on morphology and hybridization signal with reflection-contrast microscopy (RCM) and transmission electron microscopy (TEM). In chessboard experiments, a range of fixatives containing formaldehyde, glutaraldehyde, or both, and various permeabilization protocols, including ethanol and pepsin treatment, were investigated. A transfected rat fibroblast cell line that harbors an inducible human cytomegalovirus immediate early (IE) transcription unit, and specific probes for 28S ribosomal RNA and IE messenger RNA were used for this purpose. Probes were labeled with digoxigenin and hybrids were detected with anti-digoxigenin F(ab)2 fragments conjugated to horseradish peroxidase, followed by diaminobenzidine/H2O2 reaction. Effects of fixation and pre-treatments on RNA detection efficiency and morphology were monitored by RCM on whole cells. After Epon embedding and ultra-thin cross-sectioning, the corresponding TEM images were obtained. With the pre-treatments analyzed, it appeared impossible to find an acceptable balance between ISH signals and preservation of ultrastructural morphology: when good signal-to-noise ratios are obtained, the ultrastructural morphology is already deteriorated. We discuss the parameters that influence the fragile balance between high RNA detection efficiency and good preservation of ultrastructure and the benefit of RCM monitoring in the development and procedures for pre-embedding electron microscopic ISH.
Production of transferrin-like immunoreactivity by rat anterior pituitary and intermediate lobe.
Authors: Tilemans D , Vijver V V , Verhoeven G , Denef C
Published: April 2011

Using a specific anti-rat transferrin (Tf) antiserum, Tf-like immunoreactivity (Tf-lir) was detected by immunostaining in intact rat pituitaries and in reaggregated pituitary cells cultured in serum-free medium. Tf-lir cells were present in the anterior pituitary (AP), and in the intermediate (IL) and neural lobes (NL). In the AP, Tf-lir cells were oval or polygonal. An unusual topographical distribution was found. Tf-lir cells mainly occurred as dense clusters in the lateral wings. In the central part of the AP, Tf-lir was found in flattened perisinusoidal cells. Double immunostaining for Tf and the different pituitary hormones showed that Tf-lir co-localized with some gonadotrophs and somatotrophs (7% and 3% of Tf-lir cells, respectively, in typical sections). No co-localization was seen with PRL, ACTH, TSH, or alpha-MSH. The distribution of Tf-lir cells and their cell shape completely differed from that of S-100-positive cells in the AP. In the IL, clusters of large stellate Tf-lir cells were found. Again, their distribution completely differed from S-100-positive cells. In the NL, diffuse staining was found. Double immunostaining of paraffin-embedded sections of reaggregate cell cultures of the AP did not reveal any co-localization of Tf-lir with ACTH, alpha-MSH, LH, FSH, TSH, GH, or PRL. In aggregates consisting of NL + IL cells, Tf-lir was located in clusters: no co-localization with ACTH or alpha-MSH could be demonstrated. Reaggregate cell cultures of AP and NL + IL secreted Tf-lir as measured by radioimmunoassay, at least during 21 days of culture. After metabolic labeling with [35S]-methionine and immunoprecipitation of [35S]-methionine-labeled material present in the culture medium of both AP and NL + IL aggregates with anti-Tf antiserum, a 35S-labeled substance was found, which on SDS-PAGE showed an apparent M(r) of approximately 78 KD, corresponding to the M(r) of rat Tf. The present data show that a specific population of cells of rat anterior pituitary is capable of synthesizing, storing, and secreting transferrin or a substance closely related to it. Cells different from melanotrophs and S-100 cells in the IL, as well as pituicytes in the NL, also appear to produce this material. We suggest that transferrin or a transferrin-like substance may have a local role in the transport of iron or other metals or may play a role as growth factor in the three lobes of the pituitary gland.
Light and electron microscopic localization of the alpha 1-chain and the E1 and E8 domains of laminin-1 in mouse kidney using monoclonal antibodies to establish the orientation of laminin-1 within basement membranes.
Authors: Miosge N , Günther E , Heyder E , Manshausen B , Herken R
Published: April 2011

To localize the different domains of the laminin-1 molecule in tissues and gain insight into their in vivo relevance, we raised rat anti-mouse monoclonal antibodies (MAbs) against the entire molecule. Then we tested eight of the 20 clones producing anti-laminin-1 MAbs to specify their reactivity towards the alpha 1-, beta 1-, and gamma 1-chains and the elastase-cleaved fragments of the laminin-1 molecule. We found three MAbs with high titers in ELISA that showed good reactivity in embedded tissue. One of these reacted specifically against the E1 fragment, one against the E8 fragment, and one MAb detected the alpha 1-chain of laminin-1 but not the beta 1- or gamma 1-chain. All three MAbs are useful for light immunohistochemical investigations on cryosections and on paraffin-embedded material, and for ultrastructural localization of laminin-1 in LR Gold-embedded mouse tissue. Antibody staining of the E1 and E8 domains of laminin-1 revealed distinct localization of the molecule in the proximal tubule basement membranes of mouse kidney. The short arms (E1) of the laminin-1 molecule are predominantly located in the lamina lucida and the long arms (E8) are oriented towards the lamina fibroreticularis. Therefore, both MAbs are useful for studies of the orientation of the laminin-1 molecule in basement membranes. The distal tubule basement membranes did not show any distinct pattern of laminin-1 distribution. In general, the distal tubules showed the strongest reactions over the entire width of the basement membrane for all three MAbs. In contrast, the proximal tubule basement membranes showed somewhat weaker reactivity but a distinct pattern of laminin-1 distribution, with the E1 fragments oriented towards the adjacent epithelial cell surface.
Immunodetection of prostaglandin D synthase: conditions of localization in a defined subclass of primary sensory neurons.
Authors: Vesin M F , Droz B
Published: April 2011

Prostaglandin (PG) D2 is synthesized by primary sensory neurons grown in vitro. The question can be raised of whether the entire population or only a particular subpopulation of primary sensory neurons synthesizes PGD2 in vivo. To clarify this issue it was necessary to demonstrate that PGD synthase activity persists in fresh dorsal root ganglion (DRG) cryostat slices by characterizing newly formed PGD2 from [14C]-arachidonic acid, and to determine by immunocytochemistry and to identify at the ultrastructural level the neuron subpopulation expressing glutathione (GSH)-independent PGD synthase. Among the various procedures tested, the most intense, selective, and reproducible immunostaining pattern was obtained after periodate-lysine-formaldehyde fixation in phosphate buffer, permeabilization with 0.25% Triton X-100, and incubation with 10 micrograms/ml purified antibodies. Under these conditions, a subpopulation of small Class B ganglion cells was strongly immunoreactive, whereas adjacent control sections treated with absorbed antibodies or with non-immune rabbit or goat serum were unreactive. To identify the subclass of the immunoreactive small Class B neurons, immunostained vibratome slices of DRG were embedded in Epon. Ganglion cell bodies loaded with immunoprecipitates in superficially cut sections were first identified and then ultrastructurally analyzed in thin sections taken from a deeper level to obtain improved preservation of the cell architecture. This procedure enabled us to demonstrate that GSH-independent PGD synthase is accumulated in Subclass B1 primary sensory neurons.
Multiparameter in situ analysis of trophoblast cells in mixed cell populations by combined DNA and RNA in situ hybridization.
Authors: Van Wijk I J , Van Vugt J M , Könst A A , Mulders M A , Nieuwint A W , Oudejans C B
Published: April 2011

We developed a non-radioactive assay for simultaneous detection of cytoplasmic mRNA and nuclear genomic DNA in fetal trophoblast cells by sequential in situ hybridization. Trophoblast-specific mRNA is detected with a digoxigenin-labeled RNA probe complementary to HLA-G, followed by visualization through the generation of stable contrast-rich DAB/Ni complexes. Genomic target DNA is subsequently visualized in labeled cells by fluorescent in situ hybridization using biotin-labeled chromosome-specific DNA probes. Simultaneous visualization of both targets is made possible using a fluorescence microscope with FITC filter and conventional brightfield light. This method allows detection of trophoblast cells within a mixed cell population and, at the same time, analysis of chromosome anomalies in the trophoblast cells identified. For prenatal diagnosis of fetal cells enriched from maternal peripheral blood during pregnancy, this multiparameter in situ analysis of immobilized fetal trophoblast cells will be very useful.
Elimination of autofluorescence in immunofluorescence microscopy with digital image processing.
Authors: Van de Lest C H , Versteeg E M , Veerkamp J H , Van Kuppevelt T H
Published: April 2011

Autofluorescence can be a very disturbing factor in immunofluorescence microscopy. We present here a method to eliminate autofluorescence. The method is based on the fact that most autofluorescent compounds have broad-banded excitation and emission spectra, whereas specific fluorescent probes have narrow spectra. Two images are recorded and digitized, one at a wavelength exciting both the fluorescent probe and the autofluorescent molecules, and one at a wavelength exciting only the latter. Subtraction of the autofluorescence signal from the total fluorescence signal, using a self-developed computer program, results in an autofluorescence-free image. The procedure is demonstrated for elimination of elastin-derived autofluorescence in human lung alveoli and for elimination of lipofuscin-derived autofluorescence in human heart muscle. The autofluorescence signal is positively correlated with tissue section thickness (r = 0.93; p < 0.0001), and can be used to correct the specific fluorescence signals for section thickness.
Myofibrillar ATPase activity of feline muscle fibers expressing slow and fast myosin heavy chains.
Authors: Talmadge R J , Roy R R , Jiang B , Edgerton V R
Published: April 2011

The interrelationships among myofibrillar ATPase activity (Quant-mATPase), qualitative myofibrillar ATPase staining after acid (Acid-mATPase) and alkaline (Alk-mATPase) preincubations, and myosin heavy chain (MHC) composition were determined in frozen sections of soleus (Sol) and medial gastrocnemius (MG) muscle fibers from adult control cats and cats 6 months after complete spinal cord transection (Sp). Fibers were categorized as either fast, slow, or fast and slow (Fast-Slow) based on monoclonal antibody labeling. Slow fibers had low Quant-mATPase activity and stained lightly with Alk-mATPase and darkly with Acid-mATPase. Fast fibers had high Quant-mATPase activity (approximately twice that of slow fibers) and stained darkly with Alk-mATPase and lightly with Acid-mATPase. Fast-Slow fibers had intermediate Quant-mATPase activity and stained intermediately for Acid-mATPase and darkly for Alk-mATPase. There was a positive linear relationship between Alk-mATPase and Quant-mATPase for all fibers of Sol and MG from control and Sp cats. There was a negative linear relationship between Acid-mATPase and Quant-mATPase for all fibers of Sol and MG. However, within the fast fiber population of the MG there was a positive relationship between these two measures of mATPase. In summary, quantitative and qualitative measures of mATPase are highly correlated with the types of MHC expressed by single fibers from control and Sp cat muscles.
Non-radioactive localization of substance P binding sites in rat brain and spinal cord using peptides labeled with 1.4-nm gold particles.
Authors: Segond von Banchet G , Heppelmann B
Published: April 2011

We present here a new method for non-radioactive labeling of substance P (SP) to demonstrate the distribution of its binding sites in histological sections. The peptide was labeled at the primary amino group with a 1.4-nm gold particle. In Western blots of membrane fractions of rat spinal cord, specific binding occurred at 38 and 58 KD. This binding was competitively suppressed by adding the native peptide. In addition, the SP-gold conjugate was able to displace the corresponding 125I-labeled peptide from binding proteins. In histological sections, binding sites could be shown in various parts of rat brain and spinal cord. The distribution patterns were comparable to those found in studies using autoradiographic methods. Adding the native peptide or a neurokinin 1 receptor agonist markedly reduced labeling of the tissue, whereas only a slight reduction was obtained after adding neurokinin A. Therefore, SP could be specifically labeled with a 1.4-nm gold particle to demonstrate its binding sites. This new method combines the advantages of receptor-ligand affinity binding with a non-radioactive detection system. It can be used for labeling peptides in general and therefore offers an alternative or addition to other methods used in study of the distribution of membrane receptors.
Expression and localization of mRNA for the 16 KD subunit of V-ATPase in the rat embryo.
Authors: Numata M , Ohkuma S , Iseki S
Published: April 2011

Vacuolar H(+)-ATPase (V-ATPase), an enzyme composed of multisubunits, is located in the membrane of intracellular organelles (e.g., lysosomes, and endosomes) and maintains the intraorganellar acidic pH by pumping protons across the membrane. Although there is growing evidence for some important role of V-ATPase in cell proliferation and differentiation, the functional significance of V-ATPase in vivo during mammalian development remains obscure. In the present study we investigated the expression and localization of mRNA for the 16 KD subunit of V-ATPase, an essential sector for enzymatic activity, in prenatal rat by Northern blot analysis and in situ hybridization with a specific oligonucleotide probe. With Northern blot analysis, consistent expression of the mRNA was observed in the embryos throughout the period examined (E14-E20). On in situ hybridization, mRNA signal was distributed with various intensities in both the epithelial and mesenchymal tissues at embryonic day 14 (E14). In E17 and E20 embryos, localization of strong signal became more restricted to distinct mesenchymal cells such as fibroblasts adjacent to the epithelia of skin, lung, and intestine, the cells of perichondrium, and myoblasts in the process of fusion. These results suggest that V-ATPase performs specific functions during the later stages of embryogenesis, especially at sites of mesenchymal differentiation and epithelium-mesenchyme interaction.
Immunocytochemical investigation of insulin secretion by pancreatic beta-cells in control and diabetic Psammomys obesus.
Authors: Bendayan M , Malide D , Ziv E , Levy E , Ben-Sasson R , Kalman R , Bar-On H , Chrétien M , Seidah N
Published: April 2011

Hyperproinsulinemia is a characteristic feature of non-insulin-dependent diabetes mellitus (NIDDM) caused by pancreatic beta-cell dysfunction through a secretion-related alteration or impaired proinsulin processing. We have investigated the insulin processing and secretion in Psammomys obesus fed with low- and high-energy diets, which represent a model for diet-induced NIDDM. With a high-energy diet the animals develop hyperglycemia and hyperinsulinemia, whereas those maintained on a low-energy diet remain normoglycemic. Although a large amount of insulin immunoreactivity was detected in beta-cells of the normoglycemic compared to hyperglycemic animals, in situ hybridization for insulin mRNA demonstrated a particularly high signal in the beta-cells of the hyperglycemic animals. By electron microscopy, the beta-cells of normoglycemic animals displayed large accumulations of secretory granules, whereas those of the hyperglycemic animals contained very few granules and large deposits of glycogen. These results reflect a secretory resting condition for the cells of the normoglycemic animals in contrast to stimulated synthetic and secretory activities in the cells of the hyperglycemic ones. Using colloidal gold immunocytochemistry at the electron microscopic level, we have examined subcellular proinsulin processing in relation to the convertases PC1 and PC2. Immunolabeling of proinsulin, insulin, C-peptide, PC1, and PC2 in different cell compartments involved in beta-cell secretion were evaluated. Both PC1 and PC2 antigenic sites were detected in beta-cells of hyperglycemic Psammomys, but their labeling intensity was weak compared to the cells of normoglycemic animals. In both groups of animals, higher levels of PC2 were found in the Golgi apparatus than in the immature granules. Major decreases in proinsulin, insulin, PC1, and PC2 immunoreactivity were recorded in beta-cells of the hyperglycemic Psammomys. In addition, all these antigenic sites were detected in lysosome-like structures, revealing a major degradation process. These results suggest that the insulin-secreting cells in hyperglycemic Psammomys obesus are in a chronic secretory state during which impaired processing of proinsulin appears to take place.
Co-localization of manganese superoxide dismutase and NADH diaphorase.
Authors: Fang S , Thomas R M , Conklin J L , Oberley L W , Christensen J
Published: April 2011

Manganese superoxide dismutase (Mn SOD), mitochondrial enzyme, defends against the toxic effects of superoxide radical (O2.-) in pathological processes by catalyzing the conversion of O2.- to hydrogen peroxide (H2O2). The activity of another enzyme, NADH diaphorase, forms the basis for a histochemical method used commonly to demonstrate nerve cell bodies in the enteric plexuses. We found identical patterns of localization of Mn SOD immunoreactivity and NADH diaphorase activity in brain, esophagus, stomach, colon, liver, and kidney. NADH diaphorase enzymatic activity co-migrated with complexes of Mn SOD on a non-denaturing gel. This suggests that the NADH diaphorase may in some way be related to Mn SOD.
Effects of fixation and paraffin embedding on the immunohistological detection of cell-associated HIV-1 by different monoclonal antibodies.
Authors: Pudney J , Anderson D
Published: April 2011

This study evaluates a panel of monoclonal antibodies (MAbs) to HIV-1 antigens (DuPont anti-gp120, gp41, p24; Olympus anti-gp120/160, gp41, p24A, p24B, p55, p18A, p18B, reverse transcriptase) for their ability to detect the virus in tissues after exposure to various fixatives (100% acetone, 10% formaldehyde, 2.5% glutaraldehyde, 4% paraformaldehyde/1% glutaraldehyde, Bouin's fluid) and after paraffin embedding. Acetone, 10% formaldehyde, and Bouin's fluid all preserved a wide range of viral epitopes compared with other fixatives. The most robust MAbs were DuPont p24 and Olympus p55, which produced excellent staining regardless of the fixative used. Embedding in paraffin variability influenced the capacity of MAbs to detect HIV-1 epitopes on fixed cells. Certain antibodies (e.g., DuPont gp24, Olympus p24B) produced good staining, whereas other epitopes (e.g., DuPont gp120, formaldehyde) were destroyed. In some cases, paraffin embedding revealed antigenic sites that had been formerly masked (e.g., Olympus gp120 and p24A; formaldehyde and glutaraldehyde fixation). These results indicate that HIV-1 antigens can be detected by immunohistology on cells exposed to most common fixatives. Therefore, retrospective analysis of pathological material is possible, provided that the antibodies are matched to the fixative used to preserve the tissue.
Fluorescent conjugates of brefeldin A selectively stain the endoplasmic reticulum and Golgi complex of living cells.
Authors: Deng Y , Bennink J R , Kang H C , Haugland R P , Yewdell J W
Published: April 2011

The fungal metabolite brefeldin A (BFA) interferes with vesicular trafficking in most animal cells. To gain insight into the mechanism of BFA action, we esterified it to the fluorophore, boron dipyromethene difluoride (BODIPY). BODIPY-BEA localized predominantly in the endoplasmic reticulum (ER) and Golgi complex of viable cells and was extracted by detergent treatment, suggesting it interacts primarily with lipid bilayers. The localization of the conjugate is conferred by BFA, since free BODIPY or BODIPY esterified to cyclopentanol did not specifically localize to internal membranes. BODIPY-BFA exhibited a similar biological activity to BFA, but only when used at higher concentrations and after a delay. HPLC analysis revealed that over this period, cells converted BODIPY-BFA to species co-eluting with free BODIPY and BFA. Therefore, BODIPY-BFA is probably inactive until BFA is released by cellular esterases. The specific localization of BODIPY-BFA to the ER and Golgi complex suggests that BFA might exert its effects on vesicular trafficking by perturbing the lipid bilayer of its target organelles. Because BODIPY-BFA intensely stains the ER at concentrations that have no discernible effects on intracellular transport or other cellular functions, it should be useful for visualizing the ER in living cells.
High-sensitivity diagnosis of AA amyloidosis using Congo red and immunohistochemistry detects missed amyloid deposits.
Authors: Linke R P , Gärtner H V , Michels H
Published: April 2011

Biopsy diagnosis of early amyloid-A (AA) amyloidosis has often been difficult. Examination of 57 consecutive biopsy specimens from 42 patients with inflammatory pediatric diseases permitted comparison of the precision of biopsy amyloid diagnosis in six different laboratories (labs), which applied the following methods: Congo red alone (four unspecialized labs combined as Lab 1), Congo red and electron microscopy (Lab 2), or Congo red and immunohistochemistry using monoclonal antibodies (Lab 3). Lab 3 reexamined the diagnoses made by Lab 1 and Lab 2. Of the 42 patients, 17 patients with 32 biopsies were selected for this study based on the presence of amyloid in at least one biopsy. Whereas massive or no amyloid was concordantly recognized by all labs in 18 biopsies from nine patients, discordance was demonstrated in 14 biopsies from eight patients. Comparison of Labs 1-3 revealed amyloid in 12 rectal and 18 renal biopsies evaluated by Lab 3, whereas Lab 2 missed amyloid in two of 18 renal biopsies and Lab 1 missed amyloid in 11 of 12 rectal biopsies. Most amyloid was missed when only minute amounts of amyloid were present. Had our technique (Lab 3) been available at the time of biopsy, amyloid could have been diagnosed years earlier, thereby sparing the patient further biopsies and allowing initiation of earlier treatment before organ damage could occur.
Amount variability of total and individual Ag-NOR proteins in cells stimulated to proliferate.
Authors: Sirri V , Roussel P , Trerè D , Derenzini M , Hernandez-Verdun D
Published: April 2011

Ribosomal genes are associated with a subset of acidic proteins called Ag-NOR proteins. The amount of nucleolar Ag-NOR proteins varies, depending on nucleolar activity and/or cell proliferation. To understand the linkage between the amount of Ag-NOR proteins, ribosome biogenesis, and cell proliferation, we investigated the variability of Ag-NOR proteins in rRNA-stimulated cells maintained in G1 and in rRNA-stimulated cells entering the mitotic cycle. Rat hepatocytes were stimulated with cortisol for rRNA synthesis (1, 4, and 8 hr) and the cell cycle was induced by hepatectomy in regenerating hepatocytes (3-21 hr). In non-stimulated hepatocytes, nucleolin and protein B23 were the two major Ag-NOR proteins, corresponding to 70% of total Ag-NOR staining. In hepatocytes stimulated for rRNA synthesis in G1, the amount of Ag-NOR proteins was only slightly increased, whereas in cycle-stimulated cells it was increased 3.04-fold. This is the consequence of a differential increase of the major Ag-NOR proteins that appears earlier and is proportionally more important for nucleolin (3.5-fold) than for protein B23 (twofold) and also for the increase of several minor Ag-NOR proteins. We conclude that, in dividing cells, the mean value of the Ag-NOR proteins measured reflects the percentage of cells in the different phases. This could explain why the amount of Ag-NOR proteins can be used as a marker of cell proliferation.
Expression of plasminogen activator-inhibitor-1 (PAI-1) during cellular remodeling in proliferative glomerulonephritis in the rat.
Authors: Barnes J L , Mitchell R J , Torres E S
Published: April 2011

Pericellular proteolysis involves the plasminogen activator/plasmin system and plays an important role in cell remodeling involving cell migration and extracellular matrix turnover. Studies in this laboratory have previously characterized a model of proliferative glomerulonephritis induced by Habu snake venom (HSV) in the rat that involves cell migration, proliferation, and extracellular matrix accumulation. Because plasminogen activator-inhibitor-1 (PAI-1) has been used as a marker for cell migration as well as matrix accumulation, we were interested in examining the temporal and spatial expression and cellular sources of PAI-1 mRNA and translated protein over the course of HSV-induced proliferative glomerulonephritis. The results showed a highly localized and progressive expression of PAI-1 mRNA and translated protein by in situ hybridization and immunohistochemistry at the margins and periphery of glomerular lesions 8 and 24 hr after HSV. The expression of PAI-1 in glomerular lesions localized to the same sites as mesangial cell marker proteins, desmin and Thy-1.1, indicating that mesangial cells synthesize this important regulator proteolysis. Few cells expressed PAI-1 in the central aspects of glomerular lesions at later time intervals (48 and 72 hr) when cell proliferation and expression of extracellular matrix (fibronectin protein and mRNA) were maximal. Therefore, the expression of PAI-1 in this model was associated more with early events related to cell migration than with proliferation or extracellular matrix synthesis. These observations support the hypothesis that the plasminogen activator/plasmin system is involved in cell migration in early remodeling during glomerular disease.
Ultrastructural localization of alkaline phosphatase activity in human eccrine and apocrine sweat glands.
Authors: Saga K , Morimoto Y
Published: April 2011

Alkaline phosphatase (ALP) is a membrane-bound enzyme that catalyzes the hydrolysis of inorganic and organic monophosphate esters at alkaline pH. Although the functions of ALP are poorly understood, it is believed to be involved in membrane transport. Because little is known about the functions and distribution of ALP in the sweat glands, we studied the localization of ALP in human sweat glands with light and electron microscopic enzyme cytochemistry. In eccrine sweat glands, ALP was restricted to the cell membranes of intercellular canaliculi. Luminal cell membranes of secretory cells that are in continuity with intercellular canaliculi did not show ALP activity. These results suggest that ALP participates in the production of primary sweat at intercellular canaliculi. In apocrine sweat glands, basal cell membranes of secretory cells and myoepithelial cell membranes that were in apposition with each other showed ALP activity, where as no activity was seen in eccrine sweat glands. These differences in the distribution of ALP in myoepithelial cells between eccrine and apocrine sweat glands might be related to the functional differences of these sweat glands. ALP histochemistry could help to diagnose and to determine the direction of differentiation in sweat gland tumors.