bluuebunny/crossref_metadata_2025 huggingface dataset:
Metal Sandwich Method to Quick-freeze Monolayer Cultured Cells for Freeze-fracture
Authors: Fujimoto Toyoshi , Fujimoto Kazushi
Published: April 2011

We describe a simple quick-freezing method to obtain a large fractured plane of the plasma membrane from monolayer cultured cells. Cells were grown on thin gold foil, inverted on a thin layer of gelatin on thin copper foil, and frozen by a quick press between two gold-plated copper blocks precooled in liquid nitrogen. The frozen cell sandwich was mounted on the cold stage of a freeze-fracture device with the gold side up and was fractured by separating the sandwich with a cold fracture knife. When this technique was applied to confluent monolayer cells, large replicas of the E-face of the upper plasma membrane and the P-face of the lower plasma membrane were obtained. The present metal sandwich method is simple, does not require any expensive equipment, and provides a large fracture plane of the plasma membrane for subsequent histochemical manipulation.
Enhanced Immunohistochemical Detection of Autonomic Nerve Fibers, Cytokines and Inducible Nitric Oxide Synthase by Light and Fluorescent Microscopy in Rat Spleen
Authors: Meltzer Jon C. , Grimm Paul C. , Greenberg Arnold H. , Nance Dwight M.
Published: April 2011

We have developed enhanced immunohistochemical protocols for detecting autonomic nerve fibers and splenocyte-associated proteins in rat spleen. This includes norepinephrine-synthesizing enzymes (dopamine-βeta hydroxylase (DBH) and tyrosine hydroxylase (TH)), neuropeptide Y (NPY), tumor necrosis factor -α (TNF-α), interferon-γ (IFN-γ), c-fos protein, inducible nitric oxide synthase (iNOS), and the macrophage cell marker ED1. Animals were divided into sham-operated and splenic nerve-sectioned groups for detection of DBH, TH, and NPY. For immunodetection of TNF-α, iNOS, IFN-γ and c-fos, animals were injected IV with saline or 100 μg of lipopolysaccharide (LPS) and were sacrificed at various time intervals post injection. Rats were perfused with 4% paraformaldehyde, spleens removed and cryoprotected, and 50-μm floating sections were cut on a freezing microtome. Immunodetection was performed with various detection systems and substrate/chromogen solutions, and in some cases using pretreatment with proteinase K (PK) for antigen unmasking. PK pretreatment increased immunostaining for DBH, TH, NPY, IFN-γ, iNOS, and ED1, and the improvement was concentration-dependent. Using NPY immunostaining to index the signal-to-noise ratio for various substrates and detection systems, we found that an alkaline phosphatase detection system with NBT/BCIP as a substrate was the best procedure for light microscopy, whereas the CY3-labeled secondary antibody technique proved optimal for fluorescent microscopy. Surgical transection of the splenic nerve eliminated all nerve fiber staining for DBH, TH, and NPY. TNF-α, IFN-γ, c-fos, and iNOS proteins were observed in the spleen in a time-dependent manner after LPS stimulation. Fluorescent double labeling, visualized with fluorescent confocal scanning laser microscopy, revealed many NPY fibers distributed among the ED1-labeled macrophages. These results demonstrate that immunohistochemistry can be used to index the activational effects of an immune challenge on splenocytes in situ and verifies that splenic immune cells are innervated by the sympathetic nervous system.
Apoptosis and Proliferation of Fibroblasts During Postnatal Skin Development and Scleroderma in the Tight-skin Mouse
Authors: Pablos José L. , Carreira Patricia E. , Serrano Lourdes , Castillo Pedro Del , Gomez-Reino Juan J.
Published: April 2011

Tight-skin (Tsk) is a dominant gene mutation that causes a fibrotic skin disease in mice, similar to human scleroderma. Both conditions are characterized by increased numbers of dermal fibroblasts containing high levels of procollagen mRNA. Whether this fibroblast population arises from fibroblast growth or fibroblast transcriptional activation is debated. Proliferation and apoptosis of fibroblasts of normal and Tsk mice were studied in skin sections before, at onset, and in established fibrosis. Tissue sections were immunostained with proliferating cell nuclear antigen (PCNA) as proliferation marker. Apoptosis was investigated by in situ end-labeling of fragmented DNA and nuclear staining with propidium iodide. The expression of the apoptosis inhibitor Bcl-2 was investigated by immuno-histochemistry. We demonstrate differences in fibroblast proliferation and apoptosis related to postnatal skin growth and development. Neonatal skin exhibits the highest levels of proliferation and apoptosis in fibroblasts. In contrast, low proliferation and absence of apoptosis characterizes adult fibroblasts. Skin fibroblasts express Bcl-2 only in newborns, and at other ages Bcl-2 was restricted to epithelial cells. Our results also suggest that neither increased fibroblast proliferation nor defective apoptosis accounts for the fibrotic phenotype of Tsk. Therefore, transcriptional activation of extracellular matrix genes appears more relevant in the pathogenesis of Tsk fibrosis.
Detection of Integrins in Formalin-fixed, Paraffin-embedded Tissues
Authors: Liapis Helen , Hutton Karen
Published: April 2011

Integrins are heterodimeric transmembrane receptors, which are expressed in many cells. In vitro experiments have demonstrated that integrins may be important in tumor progression and organ development. The functions of integrins were previously studied in cell cultures and their tissue expression was detected by immunofluorescence or immunoperoxidase in frozen sections. The purpose of this study was to determine the optimal conditions for detection of integrins in formalin-fixed, paraffin-embedded tissues. We utilized microwave heating and enzyme digestion in routinely processed, surgically removed tissues. Our results demonstrate that integrins can be reliably detected in archival material. This approach will facilitate further investigation of the role played by integrins in human malignancies and in developmental processes.
A Novel Technique For Mapping the Lipid Composition of Atherosclerotic Fatty Streaks by En Face Fluorescence Microscopy
Authors: Klinkner Anne M. , Bugelski Peter J. , Waites C. Robbie , Louden Calvert , Hart Timothy K. , Kerns William D.
Published: April 2011

We introduce here a new fluorescence microscopy technique for en face analysis of the atherosclerotic fatty streaks (FS). This technique is semiquantitative and has the sensitivity and resolution to map lipids to individual cells in FS less than 100 μm in diameter. New Zealand White rabbits were fed an atherogenic diet for up to 26 weeks. Aortas were fixed in formalin and stained en bloc with the fluorescent dyes Nile red and filipin. Fluorescent staining was validated by correlating microfluorimetric and biochemical measurements of the lipid content in FS. To determine the cell types associated with the different staining patterns, FS were also evaluated by transmission electron microscopy (TEM) and immunohistochemistry (IH). Correlation of microfluorimetry, TEM, IH, and biochemical data indicated that regions rich in non-esterified cholesterol stained with filipin and fluoresced blue owing to accumulations of lipid vessicles and/or cholesterol crystals. Regions rich in neutral and polar lipids stained with Nile red and fluoresced yellow or orange, respectively, owing to accumulations of lipids in both macrophages and smooth muscle cells (SMC). Digital overlays of the filipin and Nile red images revealed that larger lesions (>0.5 mm diameter) had a “nested” distribution of lipids, with a blue (filipin) fringe surrounding an orange (Nile red) fringe surrounding a yellow (Nile red) center.
Dde-I Restriction Endonuclease Fragmentation: A Novel Method of Generating cDNA Probes for In Situ Hybridization in Brain
Authors: Melloni Richard H. , Aronin Neil , DeGennaro Louis J. , Ferris Craig F. , Harrison Robert J.
Published: April 2011

We present a novel procedure for detection of low- and high-abundance messenger RNAs in the brain by in situ hybridization histochemistry, by using fragmented double-stranded cDNA as molecular probes. The procedure involves digesting the cDNA of interest with the restriction endonuclease from Desulfocibrio desulfuricans (Dde I digestion), followed by random primed labeling, which generates a family of high specific activity cDNA fragments. This procedure is a rapid, straightforward, and reproducible method of obtaining sensitive probes for in situ hybridization and is generally applicable to the analysis of the expression of a large number of genes. Here we report the use of this procedure to prepare probes for the detection of synapsin I, p150Glued, neurotensin, c-fos, and c-jun mRNAs in brain, using both isotopic and non-isotopic labeling methods. Because this procedure does not require complex recombinant DNA manipulations or oligonucleotide design, it should prove useful to the non-molecular biologist examining the expression of genes in the central nervous system.
Cloning and Expression of Murine SC1, a Gene Product Homologous to SPARC
Authors: Soderling Jacquelyn A. , Reed May J. , Corsa Amoreena , Sage E. Helene
Published: April 2011

A number of cDNAs (SC1, QR1, and hevin) have been shown to be similar to SPARC (secreted protein acidic and rich in cysteine), a matricellular protein that regulates cell adhesion, cell cycle, and matrix assembly and remodeling. These proteins are 61–65% identical in the final 200 residues of their C-termini; their N-terminal sequences are related but more divergent. All have an overall acidic pl, with a follistatin-like region that is rich in cysteine, and a Ca+2 binding consensus sequence at the C-terminus. Using degenerate primers representing the most highly conserved region in SPARC, SC1, and QR1, we identified a 300-bp SC1 clone in a primary polymerase chain reaction (PCR) screen of a mouse brain cDNA library. This cDNA was used to obtain a full-length clone, which hybridized to a 2.8-kb RNA abundant in brain. Mouse SC1 displays a similarity of 70% to mouse SPARC at the amino acid level. Northern blot and RNAse protection assays revealed a 2.8-kb mRNA expressed at moderate levels (relative to brain) in mouse heart, adrenal gland, epididymis, and lung, and at low levels in kidney, eye, liver, spleen, submandibular gland, and testis. In contrast to SPARC, in situ hybridization showed expression of SC1 mRNA in the tunica media and/or adventitia of medium and large vessels; transcripts were not detected in capillaries, venules, or large lymphatics. The distribution of transcripts for SC1 was also different from that of SPARC in several organs, including adrenal gland, lung, heart, liver, and spleen. Moreover, SC1 mRNA was not evident in endothelium cultured from rat heart, bovine fetal and adult aorta, mouse aorta, human omentum, and bovine retina. Cultured smooth muscle cells and fibroblasts also failed to express SC1 mRNA. The absence of SC1 transcript in cultured cells indicates that the SC1 gene is potentially sensitive to regulatory factors in serum or to a three-dimensional architecture conferred by the extracellular matrix that is lacking in vitro. In conclusion, the expression of SPARC and SC1 appears to be coincident in specific tissues (e.g., adrenal gland and brain), but these proteins exhibit distinct expression patterns in most organs of the mouse. Because SC1 and SPARC are structurally similar and exhibit counteradhesive effects on cultured cells, their overlapping and/or adjacent expression in most tissues predicts that one protein might compensate functionally, at least in part, for the other. The sequence reported here has been deposited in the GenBank data base (accession no. U77330).
Inducible Nitric Oxide Synthase in the Anterior Pituitary Gland: Induction by Interferon-γ in a Subpopulation of Folliculostellate Cells and in an Unidentifiable Population of Non-hormone-secreting Cells
Authors: Vankelecom Hugo , Matthys Patrick , Denef Carl
Published: April 2011

In the context of immune-endocrine relationships, we have previously shown that interferon-γ (IFN-γ) inhibits hormone secretion in anterior pituitary (AP) cell cultures. The non-hormone-secreting folliculostellate (FS) cells were found to mediate this inhibitory action. Because in the immune system IFN-γ is a strong stimulator of nitric oxide (NO) release through the induction of NO synthase (NOS), we investigated whether the inducible form of NOS (iNOS) is present in (rat) AP cell cultures, and whether its expression is stimulated by IFN-γ. Immunocytochemistry revealed that under basal in vitro conditions only a very few AP cells contained iNOS. Treatment with IFN-γ caused a sixfold rise in the number of iNOS-positive cells and augmented the intensity of the staining. The increased number of iNOS-expressing cells was paralleled by elevated production of NO. Some of the iNOS-positive cells extended cytoplasmic processes between hormone-secreting cells, which is a characteristic of FS cells. Immunostaining of FS cell-poor and FS cell-enriched populations (obtained by gradient sedimentation) also suggested the presence of iNOS in a subpopulation of FS cells. By double immunofluorescence techniques we found that about 65% of iNOS-expressing cells were positive for S-100, a marker protein for FS cells. However, around 80% of the S-100-positive cells were not labeled for iNOS. On the other hand, the majority of the S-100-negative iNOS-containing cells could not be further identified by antisera against the classical AP hormones, suggesting the presence of iNOS in a still unidentified non-hormone-secreting cell type of the AP gland. This report is the first to demonstrate the expression of the inducible form of NOS in the AP gland. IFN-γ upregulates this expression, showing that cytokines may use the same signaling mechanisms in both the immune and the endocrine system. In addition, a putative new function of a subpopulation of FS cells in the paracrine regulation of the AP gland is suggested.
Immunohistochemical Localization of Arginine and Citrulline in Rat Renal Tissue
Authors: Aoki Eiko , Takeuchi Ikuo K.
Published: April 2011

Using antibodies highly specific for L-arginine and L-citrulline, we localized these amino acids in rat kidney with immunohistochemical methods. Highest levels of arginine immunoreactivity were observed in epithelial cells of proximal tubules in the outer stripe of the outer medulla and the collecting ducts in the cortex. Staining intensity of proximal convoluted tubules in the outer stripe decreased from the inner side to the outer side. In the inner medulla, collecting ducts were labeled with moderate intensity. Staining within the cortex was apparent only with collecting ducts. Citrulline immunoreactivity was localized in the epithelial cells of collecting ducts both in the cortex and medulla. Immunoreactivity was also found in glomerular podocytes and in the epithelial cells of proximal convoluted tubules in the outer medulla. These localizations were different from those of other amino acids previously reported. The precise cellular distribution of arginine and citrulline in rat kidney was determined for the first time by an immunohistochemical method in the present study.
Major DNA Fragmentation Is a Late Event in Apoptosis
Authors: Collins Jae A. , Schandl Cynthia A. , Young Kristy K. , Vesely Josef , Willingham Mark C.
Published: April 2011

Apoptosis, the terminal morphological and biochemical events of programmed cell death, is characterized by specific changes in cell surface and nuclear morphology. In addition, DNA fragmentation in an internucleosomal pattern is detectable in mass cultures of apoptotic cells. However, DNA fragmentation and nuclear morphological changes may not necessarily be associated events. In this study, we examined OVCAR-3 and KB human carcinoma cells using time-lapse video phase-contrast microscopy to characterize the surface and nuclear morphological features of apoptosis in response to treatment with either taxol or ricin. The surface morphological features of apoptosis were the same in both cell types and with both drugs. Using an in situ nick-translation histochemical assay, these single cells were also examined for DNA strand breaks during apoptosis. Surface morphological changes demonstrated discrete stages of cell rounding, surface blebbing, followed by cessation of movement and the extension of thin surface microspikes, followed much later by surface blistering and cell lysis. Nuclear features examined by DAPI cytochemistry demonstrated apoptotic nuclear condensation very early in this sequence, usually at the time of initial surface blebbing. The nick-translation assay, however, demonstrated DNA strand breaks at a much later time, only after the formation of separated apoptotic bodies or after final cell lysis. This study points out the differences between surface and nuclear morphological changes in apoptosis, and the large temporal separation between nuclear morphological changes and major DNA fragmentation detectable by this in situ technique. This result suggests caution in using in situ nick-translation as a direct correlate of internucleosomal DNA fragmentation in apoptosis.
Immunolocalization of Rap1 in the Rat Parotid Gland: Detection on Secretory Granule Membranes
Authors: D'Silva Nisha J. , DiJulio Dennis H. , Belton Carol M. , Jacobson Kerry L. , Watson E.L.
Published: April 2011

The objective of this study was to localize rap1 in the rat parotid gland. Rap1 is a small GTP-binding protein that has been linked to phagocytosis in neutrophils and various functions in platelets. In this study, we used [α-32 P]-GTP-blot overlay analysis, immunoblot analysis, and immunohistochemistry to identify rap1 in rat parotid gland. The immunohistochemical techniques included immunoperoxidase and widefield microscopy with image deconvolution. Rap1 was identified in the secretory granule membrane (SGM), plasma membrane (PM), and cytosolic (CY) fractions, with the largest signal being in the SGM fraction. The tightly bound vs loosely adherent nature of SGM-associated rap1 was determined using sodium carbonate, and its orientation on whole granules was assessed by trypsin digestion. Rap1 was found to be a tightly bound protein rather than a loosely adherent contaminant protein of the SGM. Its orientation on the cytosolic face of the secretory granule (SG) is of significance in postulating a function for rap1 because exocytosis involves the fusion of the cytoplasmic face of the SG with the cytoplasmic face of the PM, with subsequent release of granule contents (CO). Therefore, the localization and high concentration of rap1 on the SGM and its cytosolic orientation suggest that it may play a role in the regulation of secretion.
Tomography of Cells by Confocal Laser Scanning Microscopy and Computer-assisted Three-dimensional Image Reconstruction: Localization of Cathepsin B in Tumor Cells Penetrating Collagen Gels In Vitro
Authors: Strohmaier Anja-Rose , Porwol Torsten , Acker Helmut , Spiess Eberhard
Published: April 2011

We used the nondestructive procedures of confocal laser scanning microscopy in combination with computer-assisted methods to visualize tumor cells in the process of penetrating collagen gels. Three independent sets of images were collected. The image information of all data sets was combined into one image, giving a three-dimensional (3D) impression at high light microscopic resolution and sensitivity. We collected information about the extracellular matrix using the reflection mode, the cell surface/morphology by staining with the fluorescent dye DiOC6(3), and the distribution of cathepsin B by Cy-3-labeled immunolocalization. The specific aim of our study was visualization of the spatial relationship of cell organelles as far as they contain the enzyme cathepsin B to cell morphology and motility in a 3D model of extracellular matrix. The majority of the enzyme was localized pericellularly, with no visible relationship to the direction of movement. However, substantial amounts also appeared in intramatrix pseudopodia and associated with the extracellular face of the plasma membane, which may be indicative either of secretion and/or epicellular activity. Our approach has general applicability to study of the spatial relationships of cell compartments and their possible reorganization over time. This could open new horizons in understanding cell structure and function.
Production and Characterization of Specific Anti-peptide Antiserum Against Free α-subunit of Rat Pituitary Glycoprotein Hormones
Authors: Tanaka Shigeyasu , Kurabuchi Shingo , Mochida Hiroshi , Hayashi Hiroaki , Wakabayashi Katsumi
Published: April 2011

To obtain an antibody specific for the α-subunit of rat pituitary glycoprotein hormones, we synthesized a peptide corresponding to the sequence 37–53 (ST-7: Phe-Ser-Arg-Ala-Tyr-Pro-Thr-Pro-Ala-Arg-Ser-Lys-Lys-Thr-Met-Leu-Val) of the rat α-subunit. The polyclonal antiserum against this peptide was generated in rabbits. This region is hydrophilic and highly conserved among several mammalian species. Noncompetitive binding tests showed that the ST-7 antiserum had specific affinity for the rat free α-subunit, but not for rat intact LH, FSH, and TSH. The ST-7 antiserum immunostained two types of cells in the rat anterior pituitary, i.e., gonadotrophs and thyrotrophs. This was also the case in mouse, cattle, sheep, and pig, which have an identical sequence of ST-7 in their α-subunit. The pituitary cells of horse (Arg substituted for Lys as residue 48 of the rat α-subunit), human, and eel (Leu for Ala at residue 45), chicken (Met for Ala at residue 45), and bullfrog (Tyr for Phe at residue 37 and Met for Ala at residue 45) were not stained with the ST-7 antiserum. This study indicated that the ST-7 antiserum is sequence-specific for the α-subunit and is therefore useful for immunohistochemical studies on the secretory pathway of the free α-subunit.
N-Ethylmaleimide (NEM) Can Significantly Improve In Situ Hybridization Results Using 35S-labeled Oligodeoxynucleotide or Complementary RNA Probes
Authors: Zoeller R. Thomas , Fletcher Donald L. , Butnariu Olimpia , Lowry Christopher A. , Moore Frank L.
Published: April 2011

We predicted that a significant source of background labeling after in situ hybridization (ISH) using 35S-labeled probes is attributable to a chemical reaction between the phosphorothioate moiety of the probe [O3 P=S] and disulfides in tissue. These covalent bonds would immobilize probe in the tissue, thereby increasing background labeling. On the basis of this view, we have explored the use of N-ethylmaleimide (NEM) to irreversibly alkylate the phosphorothioate moiety of the probe and/or to alkylate free sulfhydryls in tissue to block the formation of disulfides as a method of reducing background labeling. We report that NEM can significantly decrease background labeling of 35S-labeled oligodeoxy-nucleotide or cRNA probes but does not affect specific labeling. We conclude that the use of NEM in ISH protocols, as outlined here, may be an additional element researchers may consider to improve the signal-to-noise ratio.
Anti-biotin Antibodies Offer Superior Organelle-specific Labeling of Mitochondria over Avidin or Streptavidin
Authors: Hollinshead Michael , Sanderson Jeremy , Vaux David J.
Published: April 2011

The mitochondrial matrix contains endogenously biotinylated proteins. These proteins can cause unexpected background signal when biotin–avidin- or biotin–streptavidin-based detection systems are used in immunocytochemistry. Here we show that this reactivity can be deliberately exploited, using a simple anti-biotin reagent, to obtain strong and highly specific labeling of mitochondria by both light and electron microscopy. The signal is substantially stronger than when either avidin or streptavidin is used to detect the endogenous biotin. These results confirm the accessibility of protein-bound endogenous biotin to exogenous probes, and localize the biotinylated enzymes to the mitochondrial matrix. (J Histochem Cytochem 45:1053–1057, 1997)
Expression of Human Group II Phospholipase A2 in Transgenic Mice
Authors: Nevalainen Timo J. , Laine V. Jukka O. , Grass David S.
Published: April 2011

Group II phospholipase A2 (PLA2) has been proposed to play an important role in inflammation and defense against bacterial infection. We investigated tissues of transgenic mice expressing the human group II PLA2 gene by immunohistochemistry using rabbit anti-human group II PLA2 antibodies, and by in situ hybridization by probing with human group II PLA2 mRNA anti-sense (test) and sense (control) riboprobes. By immunohis-tochemistry, human group II PLA2 was found in various mouse tissues and cell types including hepatocytes, proximal tubule cells of the kidney, epithelial cells of the renal pelvis, urinary bladder and ureter, granulosa cells of Graafian follicles, aortic intima and media, cartilage, epiphyseal bone, bronchial epithelial cells, and connective tissue cells in the dermis. By in situ hybridization, group II PLA2 mRNA was localized in hepatocytes, epidermal cells, dermal cells, connective tissue fibroblasts, epithelial and smooth muscle cells of the urinary bladder, and cells of Bowman's capsule. These results show that human group II PLA2 is expressed in large amounts in hepatocytes and many extrahepatic tissues of the transgenic mice. These animals provide a useful new tool for studies on the metabolism, in vivo effects, and physiological and pathological roles of phospholipase A2.

(J Histochem Cytochem 45:1109–1119, 1997)
Pre-osteoblastic Proliferation Assessed with BrdU in Undecalcified, Epon-embedded Adult Rat Trabecular Bone
Authors: Barou Odile , Laroche Norbert , Palle Sabine , Alexandre Christian , Lafage–Proust Marie-Hélène
Published: April 2011

We evaluated bromodeoxyuridine (BrdU) immunohistochemistry in undecal-cified adult rat tibiae to study cell kinetics in various bone compartments: primary and secondary spongiosae, periosteum, and bone marrow. Several regimens of BrdU administration were tested (IP injections and osmotic minipumps). We compared LR White resin, methylmethacrylate, and Epon–araldite embedding, microwave irradiation for antigen retrieval, several concentrations of sodium ethoxide for deplastification, and various DNA de-naturation procedures. Paraffin-embedded decalcified tibiae and Epon-embedded bowel were used as positive controls. The best results were obtained in rats labeled with 40 mg of BrdU for 72 hr using osmotic minipumps. The procedure using a Microprobe system in Epon-embedded bone tissue with a sodium ethoxide concentration of 50% for two intervals of 20 min provided the best staining quality and tissue preservation. Labeled pre-osteoblastic cells and bone marrow cells could be counted. Epon embedding allowed preservation of tetracycline double labeling performed 1 to 5 days before sacrifice. The number of labeled pre-osteoblastic cells was correlated with the double-labeled surface area measured histomorphometrically.
In Situ Measurement of Glutamate Concentrations in the Periportal, Intermediate, and Pericentral Zones of Rat Liver
Authors: Geerts Willie J.C. , Jonker Ard , Boon Louis , Meijer Alfred J. , Charles Rob , Van Noorden Cornelis J.F. , Lamers Wouter H.
Published: April 2011

We developed a quantitative histochemical assay for measurement of local glutamate concentrations in cryostat sections of rat liver. Deamination of glutamate by glutamate dehydrogenase (GDH) was coupled to the production of formazan and formazan precipitation was used for colorimetric visualization. The method was tested and validated with gelatin model sections with known glutamate concentrations. Calibration graphs showed linear relationships with high correlation coefficients (>96%) between glutamate concentrations or section thickness and absorbance values. The method was reproducible, with a constant percentage of 60 ± 5% of glutamate being converted in gelatin model sections containing glutamate concentrations of 2 mM and higher. Glutamate concentrations were estimated in periportal, intermediate, and pericentral zones of liver lobules that contain low, intermediate, and high GDH activity, respectively. In fed adult male rat livers, periportal zones contained the highest concentrations of glutamate (∼14 mM) and intermediate and pericentral zones ∼13 and 9 mM, respectively. On starvation, glutamate concentrations increased only in the small rim of pericentral cells that express glutamine synthetae, to ∼15 mM. In livers of fetal and newborn rats, glutamate was homogeneously distributed, with a concentration of ∼5 mM. In suckling rat liver, distribution of glutamate was still homogeneous but the concentration was increased to ∼8 mM. These glutamate distribution patterns were in agreement with those detected immunohistochemically.
Cellular Distribution of the Angiogenic Factor Heparin Affin Regulatory Peptide (HARP) mRNA and Protein in the Human Mammary Gland
Authors: Ledoux Dominique , Caruelle Daniele , Sabourin Jean-Christophe , Liu Jianfeng , Crepin Michel , Barritault Denis , Courty Jose
Published: April 2011

The heparin affin regulatory peptide (HARP) growth factor, also known as pleiotrophin, is a developmentally regulated protein that displays biological functions during cell growth and differentiation. To study the physiological role of this protein, we investigated the cellular distribution of HARP mRNA and protein in the resting human mammary gland. In situ hybridization histochemistry revealed that HARP mRNA was localized in alveolar myoepithelial cells, whereas alveolar epithelial cells were negative. In the stroma, HARP mRNA was localized in endothelial cells and smooth muscle cells of blood vessels. Interestingly, HARP protein and mRNA were not always co-localized. HARP protein immunocytochemistry staining was observed in an area including both alveolar myoepithelial and epithelial cells, although epithelial cells do not express HARP transcript. In contrast, the distribution of HARP protein is parallel to that of HARP mRNA in endothelial and vascular smooth muscle cells. In the light of these results, the putative role of HARP in controlling the proliferation and/or differentiation of the different mammary cell types is proposed and discussed.
ICAM-1/LFA-1 Expression in Acute Osteodestructive Joint Lesions in Collagen-induced Arthritis in Rats
Authors: Seidel Matthias F. , Keck Rolf , Vetter Hans
Published: April 2011

Collagen-induced arthritis in rats is a widely used model of rheumatoid arthritis (RA). However, the joint immunohistopathology is less well characterized. The objective of this study was therefore to analyze whole ankle joints for markers known to mediate inflammatory mechanisms in RA. Indirect immunohistochemistry was performed on undecalcified cryostat sections for intercellular adhesion molecule-1 (ICAM-1, clone 1A 29) and leukocyte function-associated antigen-1 (LFA-1, clone WT.1) expression, for CD4+ lymphocytes (clone W3/25), B-cells (clone HIS 14), and macrophages (clone ED2). Acute, osteodestructive arthritis ( n = 8) induced with bovine collagen Type II was verified by clinical and radiological measures. LFA-1 expression was found almost exclusively at sites associated with cartilage erosion or osteodestruction. ICAM-1 was similarly expressed in the vicinity of tissue degradation but also by blood vessels in peripheral areas of joint swelling. CD4+ lymphocytes and macrophages were more ubiquitous. B-cells were infrequent. In control animals ( n = 4) ICAM-1 was expressed by synovial blood vessels. Macrophages were identified at the synovial lining. The results suggest that LFA-1 and ICAM-1 mediate important inflammatory events in this model. Similar findings in human RA synovium provide further arguments that collagen-induced arthritis in rats might be regarded as a comparable disease.
Expression of RESP18 in Peptidergic and Catecholaminergic Neurons
Authors: Darlington Daniel N. , Schiller Martin R. , Mains Richard E. , Eipper Betty A.
Published: April 2011

We examined the expression of regulated endocrine-specific protein of 18-kD (RESP18) in selected peptidergic and catecholaminergic neurons of adult rat brain. In the hypothalamic paraventricular, supraoptic, and accessory nuclei, RESP18 mRNA was highly expressed in neurons immunostained for oxytocin and vasopressin. RESP18 mRNA was also highly expressed in paraventricular nucleus neurons immunostained for corticotropin-releasing hormone, thyrotropin-releasing hormone, and somatostatin. RESP18 mRNA was expressed in POMC cells of the arcuate nucleus, in neuropeptide Y cells of the dorsal teg-mental nucleus, lateral reticular nucleus, and hippocampus, and in brainstem catechola-minergic neurons. RESP18 mRNA expression was high in all paraventricular and arcuate neurons, but RESP18 protein was detectable in the perikarya of a subset of these neurons, suggesting an important post-transcriptional component to the regulation of RESP18 expression. RESP18 antisera immunostained perikarya but not axon fibers or terminals. Sub-cellular fractionation of homogenates of several hypothalamic nuclei identified RESP18 protein in fractions enriched in endoplasmic reticulum. The presence of 22- and 24-kD RESP18 isoforms in the neural lobe of the pituitary indicated that some RESP18 protein exited the endoplasmic reticulum. The post-transcriptional regulation of RESP18 expression and localization of RESP18 protein primarily to the endoplasmic reticulum suggests that RESP18 plays a regulatory role in peptidergic neurons.
AMF-R Tubules Concentrate in a Pericentriolar Microtubule Domain After MSV Transformation of Epithelial MDCK Cells
Authors: Nabi Ivan R. , Guay Ginette , Simard Danièle
Published: April 2011

Autocrine motility factor receptor (AMF-R) is localized to an intracellular microtubule-associated membranous organelle, the AMF-R tubule. In well-spread untrans-formed MDCK epithelial cells, the microtubules originate from a broad perinuclear region and AMF-R tubules extend throughout the cytoplasm of the cells. In Moloney sarcoma virus (mos)-transformed MDCK (MSV-MDCK) cells, microtubules accumulate around the centrosome, forming a microtubule domain rich in stabilized detyrosinated microtubules. AMF-R tubules are quantitatively associated with this pericentriolar microtubule domain and the rough endoplasmic reticulum and lysosomes also co-distribute with the pericentriolar mass of microtubules. The Golgi apparatus is closely associated with the microtubule organizing center (MTOC) within the juxtanuclear mass of AMF-R tubules, and no co-localization of AMF-R tubules with the Golgi marker β-COP could be detected by confocal microscopy. After nocodazole treatment and washout, microtubule nucleation occurs exclusively at the centrosome of MSV-MDCK cells, and only after microtubule extension to the cell periphery does the microtubule cytoskeleton reorganize to generate the pericentriolar microtubule domain after 30–60 min. AMF-R tubules dispersed by nocodazole treatment concentrate in the pericentriolar region in parallel with the reorganization of the microtubule cytoskeleton. MSV transformation of epithelial MDCK cells results in the stabilization of a pericentriolar microtubule domain responsible for the concentration and polarized distribution of AMF-R tubules.
Evidence of a Tubular System for Transendothelial Transport in Arterial Capillaries of the Rete Mirabile
Authors: Bendayan Moöse , Rasio Eugenio A.
Published: April 2011

The arterial endothelial cells of the rete capillaries of the eel were examined by transmission electron microscopy on thin sections, on freeze-fracture replicas, by scanning electron microscopy, after cytochemical osmium impregnation and perfusion with peroxidase. The study revealed the existence of membrane-bound tubules and vesicles that open at both the luminal and abluminal poles of the cell and at the level of the intercellular space. The tubules are straight or present successive dilations and constrictions. They branch in various directions and intrude deeply into the cell cytoplasm, forming a complex tubular network within the cell. Immunocytochemical techniques were applied on immersion-fixed tissues and on perfusion of the capillaries with albumin and insulin. These demonstrated that the tubular–vesicular system is involved in the transport of circulating proteins. Furthermore, protein A–gold immunocytochemistry has revealed the association of actin with the membranes of this system. On the basis of these results, we suggest that the transendothelial transport of serum proteins takes place by a transcytotic process through a membrane-bound tubular–vesicular system and is equivalent to the large pore system presumed from functional studies.
Localization of Soluble Guanylate Cyclase Activity in the Guinea Pig Cochlea Suggests Involvement in Regulation of Blood Flow and Supporting Cell Physiology
Authors: Fessenden James D. , Schacht Jochen
Published: April 2011

Although the nitric oxide/cGMP pathway has many important roles in biology, studies of this system in the mammalian cochlea have focused on the first enzyme in the pathway, nitric oxide synthase (NOS). However, characterization of the NO receptor, soluble guanylate cyclase (sGC), is crucial to determine the cells targeted by NO and to develop rational hypotheses of the function of this pathway in auditory processing. In this study we characterized guinea pig cochlear sGC by determining its enzymatic activity and cellular localization. In cytosolic fractions of auditory nerve, lateral wall tissues, and co-chlear neuroepithelium, addition of NO donors resulted in three- to 15-fold increases in cGMP formation. NO-stimulated sGC activity was not detected in particulate fractions. We also localized cochlear sGC activity through immunocytochemical detection of NO-stimulated cGMP. sGC activity was detected in Hensen's and Deiters' cells of the organ of Corti, as well as in vascular pericytes surrounding small capillaries in the lateral wall tissues and sensory neuroepithelium. sGC activity was not observed in sensory cells. Using NADPH-diaphorase histochemistry, NOS was localized to pillar cells and nerve fibers underlying hair cells. These results indicate that the NO/cGMP pathway may influence diverse elements of the auditory system, including cochlear blood flow and supporting cell physiology.
Tyramine Amplification Technique in Routine Immunohistochemistry
Authors: Wasielewski Reinhard von , Mengel Michael , Gignac Suzanne , Wilkens Ludwig , Werner Martin , Georgii Axel
Published: April 2011

Signal amplification in immunohistochemistry via binding of biotinylated tyramine to proteins near the site of peroxidase-labeled antibodies is a promising new technique, but studies investigating a wide range of markers are lacking. The tyramine amplification technique (TAT) was investigated on 85 antibodies using a simple and fast protocol, and TAT results were compared to those obtained with conventional immunohistochemistry. Using TAT, most of the markers could be 5- to 50-fold further diluted and still showed identical staining results compared with standard stainings (maximal 500-fold). However, the variable reactivity of the different markers with TAT underlines the need for individual testing of every antibody to determine the optimal dilution. Some antibodies against cell adhesion molecules could be demonstrated for the first time in archival, formalin-fixed tissue sections. TAT, if carefully evaluated, offers a revolutionary improvement for modern immunostaining, either to increase sensitivity or primary antibody dilutions (cost reduction). From a methodological point of view, immunohistochemistry has not reached its limits by far and TAT is an important progressive step in this developmental process. (J Histochem Cytochem 45:1455–1459, 1997)
Localization of B-DNA and Z-DNA in Terminally Differentiating Fiber Cells in the Adult Lens
Authors: Gagna Claude E. , Lambert W. Clark , Kuo Hon-Reen , Farnsworth Patricia N.
Published: April 2011

We examined histochemically and immunohistochemically the distribution of B- and Z-DNA in the epithelium and terminally differentiating dog lens fiber cells. On the basis of anti-DNA antibody reactivity, qualitative and quantitative data on B- and Z-DNA in cells were determined. Anti-B-DNA immunoreactivity gradually declined throughout nucleated fibers, with a precipitous decrease at ∼90 μm. Anti-Z-DNA antibody binding decreased with a sudden loss of immunoreactivity at ∼90 μm. The pattern of anti-B- and Z-DNA staining correlates with the loss of α-crystallin immunoreactivity, the major lens crystallin, and decreased eosin staining of proteins. Germinative zone cell nuclei showed the highest DNA probe binding values, followed by the superficial fibers, central zone, middle fibers, and deep fibers. The presence of single-stranded (ss)DNA in deeper fibers was detected by anti-ss-DNA antibodies. This is indicative of DNA degradation. These observations suggest that a dramatic reorganization of lens fiber cells' supramolecular order occurs at ∼90 μm, the phase transition zone. (J Histochem Cytochem 45:1511–1521, 1997)
Changes in Morphology and Spatial Position of Coiled Bodies during NGF-induced Neuronal Differentiation of PC12 Cells
Authors: Janevski John , Park Paul C. , Boni Umberto De
Published: April 2011

Interphase nuclei are organized into structural and functional domains. The coiled body, a nuclear organelle of unknown function, exhibits cell type-specific changes in number and morphology. Its association with nucleoli and with small nuclear ribonucleo-proteins (snRNPs) indicates that it functions in RNA processing. In cycling cells, coiled bodies are round structures not associated with nucleoli. In contrast, in neurons, they frequently present as nucleolar “caps.” To test the hypothesis that neuronal differentiation is accompanied by changes in the spatial association of coiled bodies with nucleoli and in their morphology, PC12 cells were differentiated into a neuronal phenotype with nerve growth factor (NGF) and coiled bodies detected by immunocytochemical localization of p80-coilin and snRNPs. The fraction of cells that showed coiled bodies as nucleolar caps increased from 1.6 ± 0.9% (mean ± SEM) in controls to 16.5 ± 1.6% in NGF-differentiated cultures. The fraction of cells with ring-like coiled bodies increased from 17.2 ± 5.0% in controls to 57.8 ± 4.4% in differentiated cells. This was accompanied by a decrease, from 81.2 ± 5.7% to 25.7 ± 3.1%, in the fraction of cells with small, round coiled bodies. SnRNPs remained associated with typical coiled bodies and with ring-like coiled bodies during NGF-induced recruitment of snRNPs to the nuclear periphery. Together with the observation that coiled bodies are also present as nucleolar caps in sensory neurons, the results indicate that coiled bodies alter their morphology and increase their association with nucleoli during NGF-induced neuronal differentiation. (J Histochem Cytochem 45:1523–1531, 1997)
Production of a Monoclonal Antibody by In Vitro Immunization That Recognizes a Native Chondroitin Sulfate Epitope in the Embryonic Chick Limb and Heart
Authors: Capehart Anthony A. , Wienecke Matthew M. , Kitten Gregory T. , Solursh Michael , Krug Edward L.
Published: April 2011

We report the production of a monoclonal antibody (d1C4) by in vitro immunization that has immunoreactivity with a native chondroitin sulfate epitope in embryonic chick limb and heart. Murine lymphocytes were stimulated by direct exposure to unfixed, unsolubilized precartilage mesenchymal aggregates in high-density micromass culture derived from Stage 22–23 chick limb buds. Specificity of d1C4 reactivity was demonstrated by sensitivity of immunohistochemical staining to pretreatment with chondroitinase ABC or AC, preferential immunoreactivity with chondroitin-6-sulfate glycosaminoglycan (CS-C GAG) in ELISA, and competition of immunohistochemical staining with CS-C GAG. Immunohistochemical analysis of the expression of the d1C4 epitope revealed a striking localization of immunoreactivity in the extracellular matrix (ECM) of precartilage aggregates of chick limb mesenchyme in high-density micromass culture by 16 hr and the prechondrogenic limb core at Stage 23 in vivo. Immunoreactivity in both cultured limb mesenchyme and the embryonic limb continued through differentiation of prechondrogenic condensations into cartilage tissue. In the developing chick heart, d1C4 staining was found throughout the ECM of atrioventricular cushion tissue by Stage 25, but was localized to mesenchyme adjacent to the myocardium in the outflow tract cushions. There was an abrupt demarcation between d1C4-reactive intracardiac mesenchyme and unreactive extracardiac mesenchyme of the dorsal mesocardium in the Stage 22 embryo. This study demonstrates the efficacy of in vitro immunization of lymphocytes for the production of MAbs to native ECM constituents, such as CS-GAGs. Immunohistochemical data utilizing d1C4 suggest that CS-GAGs bearing this epitope may be important in early morphogenetic events leading to cartilage differentiation in the limb and valvuloseptal morphogenesis in the heart.

(J Histochem Cytochem 45:1567–1581, 1997)
Cytochemical Studies of the Effects of Activin on Gonadotropin-releasing Hormone (GnRH) Binding by Pituitary Gonadotropes and Growth Hormone Cells
Authors: Childs Gwen V. , Unabia Geda
Published: April 2011

Activin stimulates the synthesis and secretion of follicle-stimulating hormone (FSH). It inhibits the synthesis and release of growth hormone (GH). It acts on gonadotropes by stimulating the synthesis of gonadotropin-releasing hormone (GnRH) receptors. To test activin's effects on GnRH target cells, pituitary cells from diestrous or proestrous rats were exposed to media with and without 60 ng/ml activin for 24 hr and stimulated with biotinylated GnRH (Bio-GnRH). The populations were double-labeled for Bio-GnRH and/or luteinizing hormone-β (LH-β), FSH-β, or GH antigens. In both diestrous and proestrous rats, activin stimulated more LH and FSH cells and increased the percentages of GnRH target cells. In diestrous rats, activin stimulated increases in the average area and density of Bio-GnRH label on target cells. In addition, more FSH, LH, and GH cells bound Bio-GnRH. The increment in binding by gonadotropes was not as great as that normally seen from diestrus to proestrus, suggesting that additional factors (such as estradiol) may be needed. These data suggest that activin plays an important role in the augmentation of Bio-GnRH target cells normally seen before ovulation. Its actions on GH cells may reflect a role in the transitory change from a somatotrope to a somatogonadotrope that is seen from diestrus to proestrus.