bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunolocalization of Lipoprotein(a) in Wounded Tissues
Authors: Yano Yoko , Shimokawa Kuniyasu , Okada Yume , Noma Akio
Published: April 2011

Fifty samples from inflamed tissues were examined by immunohistochemical techniques, using antibodies against apo(a), apo B, plasminogen, fibrinogen, proliferating cell nuclear antigen (PCNA), and various components of extracellular matrix. The immuno-histochemical features of granulation tissues were characterized by different stages of wound healing. In the first stage, immunoreactivities for anti-apo(a) and anti-apo B were weak and focal, whereas those for anti-plasminogen and anti-fibrinogen were strong and were widespread on the tissue surface. In the second stage, granulation tissues were covered with loose fibrous connective tissue, designated as a “fibrous cap.” In this stage, markedly positive staining for lipoprotein(a) [Lp(a)] was observed closer to the surface of the fibrous cap than plasminogen, suggesting that Lp(a) may prevent external fibrinolysis. Lp(a) was also found in endothelial cells and the extracellular space of small vessels underlying the fibrous cap. In the last stage of healing, apo(a) and apo B were not detectable in completely organized tissues. These findings suggest that Lp(a) plays a role in the wound healing.
Determination of the Localization of Gangliosides Using Anti-ganglioside Antibodies: Comparison of Fixation Methods
Authors: Schwarz Andreas , Futerman Anthony H.
Published: April 2011

Many studies have examined the localization of gangliosides using anti-ganglioside antibodies, although widely differing conclusions have been reached. We now demonstrate that the apparent localization of gangliosides can be greatly influenced by the fixation method. Using monoclonal antibody (MAb) A2B5 (which reacts with a variety of gangliosides), hippocampal neurons were labeled at the cell surface when incubated with the antibody before fixation, but when incubated after fixation the cells displayed a variety of labeling patterns, depending on the fixation method. Biochemical analysis demonstrated that some of the fixatives (particularly acetone and methanol) significantly reduced or completely depleted cellular gangliosides, implying that the immunoreactivity observed with A2B5, and with other antibodies, was not due to gangliosides. When neurons were incubated with an anti-GD1b antibody prefixation, uniform labeling of the plasma membrane was observed, but after ganglioside depletion using biochemical inhibitors of ganglioside synthesis no cell surface labeling was detected. However, even in cells depleted of gangliosides, labeling of both the cell surface and intracellular compartments was observed when the anti-GD1b antibody was applied after fixation. Moreover, after fixation, antibodies to GM4 and GD2 reacted with hippocampal neurons, although these ganglio-sides are absent from these neurons. In contrast, the JONES antibody (which reacts with 9- O-acetylated GD3) labeled neurons with a similar pattern, essentially irrespective of the fixation method. These observations demonstrate that great care must be taken in assigning gangliosides to specific cell populations or to intracellular locations solely on the basis of use of anti-ganglioside antibodies, and suggest that optimal fixation conditions must be established for each anti-ganglioside antibody.
Differential Expression of the Carbonic Anhydrase Genes for CA VII (Car7) And CA-RP VIII (Car8) in Mouse Brain
Authors: Lakkis Maha M. , O'Shea K. Sue , Tashian Richard E.
Published: April 2011

The spatial expression patterns of the two α-carbonic anhydrase genes, CA VII and CA-RP VIII (called Car7 and Car8 in the mouse) were examined in the mouse brain by in situ hybridization. These two genes are the most highly conserved evolutionarily among the mammalian α-CAs. Both genes showed a similarly wide expression pattern in the brain. In the cerebrum, mRNA expression was detected in the pia, choroid plexus, and neurons of the cortical layer, thalamus, and medial habenulae. A high level of expression appeared in the pyramidal and granular cells of the hippocampus. In the cerebellum, both Car7 and Car8 were transcribed to different degrees in the Purkinje cells, and a lower expression level occured in the molecular and granular cell layers. Transcription signals for both genes were excluded from the white matter regions.
Specificity of Antibodies Against Rodent Transforming Growth Factor-α Protein
Authors: Aoyama Hiroaki , Kato Hidenori , Dixon Darlene
Published: April 2011

We found that the immunohistochemical distribution of TGF-α varied in rodent tissues depending on the antibody used, suggesting that the specificity of anti-TGF-α antibodies differs significantly. To address this issue, we compared the specificity of two representative antibodies that have been widely used to detect rodent TGF-α. In a competition study, the antibodies were preincubated with an excess of synthetic rat TGF-α34-50 and were used for staining of rat and mouse kidneys and/or uterus. The results revealed that one of the antibodies, anti-rat TGF-α polyclonal antibody, was neutralized by the peptide, whereas the other, anti-human TGF-α monoclonal antibody, was not absorbed by the peptide up to an excess of 100-fold. Western blotting analysis showed that the anti-rat TGF-α polyclonal antibody recognized both human and rat purified TGF-α. However, the anti-human TGF-α monoclonal antibody did not detect purified rat TGF-α, although the antibody reacted with mouse proteins other than TGF-α from kidneys and uterus, purified human TGF-α, and mouse carbonic anhydrase II. These data indicate that the anti-human TGF-α monoclonal antibody does not recognize rodent TGF-α under our experimental conditions and suggest that distribution of TGF-α in rodent tissues may need to be reexamined.
Tumor Collagenase Stimulatory Factor (TCSF) Expression and Localization in Human Lung and Breast Cancers
Authors: Polette Myriam , Gilles Christine , Marchand Veronique , Lorenzato Marianne , Toole Bryan , Tournier Jean-Marie , Zucker Stanley , Birembaut Philippe
Published: April 2011

Tumor cell-derived collagenase stimulatory factor (TCSF) stimulates in vitro the biosynthesis of various matrix metalloproteinases involved in tumor invasion, such as interstitial collagenase, gelatinase A, and stromelysin 1. The expression of TCSF mRNAs was studied in vivo, using in situ hybridization and Northern blotting analysis, in seven normal tissues and in 22 squamous cell carcinomas of the lung, and in seven benign proliferations and in 22 ductal carcinomas of the mammary gland. By in situ hybridization, TCSF mRNAs were detected in 40 of 44 carcinomas, in pre-invasive and invasive cancer cells of both lung and breast cancers. TCSF mRNAs and gelatinase A mRNAs were both visualized in the same areas in serial sections in breast cancers, and were expressed by different cells, tumor cells, and fibroblasts. The histological results were confirmed by Northern blot analysis, which showed a higher expression of TCSF mRNAs in cancers than in benign and normal tissues. These observations support the hypothesis that TCSF is an important factor in lung and breast tumor progression.
Sensitive Chemiluminescence In Situ Hybridization for the Detection of Human Papillomavirus Genomes in Biopsy Specimens
Authors: Musiani Monica , Zerbini Marialuisa , Venturoli Simona , Gentilomi Giovanna , Gallinella Giorgio , Manaresi Elisabetta , Placa Michelangelo La , D'Antuono Antonietta , Roda Aldo , Pasini Patrizia
Published: April 2011

We developed a sensitive chemiluminescence in situ hybridization assay for detection of human papillomavirus (HPV) DNA for objective and semiquantitative evaluation of the results. The hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes, visualized with alkaline phosphatase as the revealing enzyme and a highly sensitive 1,2 dioxetane phosphate as chemiluminescent substrate. The light emitted from the hybridized probes was detected, analyzed, and measured using a high-performance, low light-level imaging luminograph connected to an optical microscope and to a personal computer for quantification of the photon fluxes and for image analysis. The system operated in consecutive steps: First, hybridized specimens were recorded in transmitted light. Then the net luminescent signal was recorded, and then an overlay of the two images provided by the transmitted light and by the luminescent signal allowed the spatial distribution of the target DNA to be localized, measured, and evaluated. Biopsy specimens from different pathological conditions associated with HPV, which had previously been proved positive for HPV DNA with the polymerase chain reaction (PCR), were analysed. The chemiluminescence in situ hybridization proved sensitive and specific with digoxigenin-, biotin-, or fluorescein-labeled probes, and provided an objective evaluation of the results. The results obtained with chemiluminescence in situ hybridization were also compared with results obtained with in situ hybridization with colorimetric detection, with good concordance of the data. Chemiluminescence in situ hybridization therefore offers the possibility of detecting HPV DNA with great sensitivity in biopsy specimens. Moreover, the images of the samples, stored in the computer, are a permanent record of the reaction and can also be sent for evaluation or comparison to other laboratories using computer networks.
Immunohistochemical Localization of the Na-K-Cl Co-transporter (NKCC1) in the Gerbil Inner Ear
Authors: Crouch James J. , Sakaguchi Nobuki , Lytle Christian , Schulte Bradley A.
Published: April 2011

We mapped the cellular and subcellular distribution of the Na-K-Cl co-transporter (NKCC) in the adult gerbil inner ear by immunostaining with a monoclonal antibody (MAb T4) generated against human colon NKCC. Heavy immunolabeling was seen in the basolateral plasma membrane of marginal cells in the stria vascularis and dark cells in the vestibular system. Subpopulations of fibrocytes in the cochlear spiral ligament and limbus and underlying the vestibular neurosensory epithelium also stained with moderate to strong intensity, apparently along their entire plasmalemma. Because MAb T4 recognizes both the basolateral secretory (NKCC1) and the apical absorptive (NKCC2) isoforms of the co-transporter, we employed reverse transcription and the polymerase chain reaction (RT-PCR) to explore isoform diversity in inner ear tissues. Using NKCC1 and NKCC2 isoform-specific PCR primers based on mouse and human sequences, only transcripts for NKCC1 were detected in the gerbil inner ear. The presence of abundant NKCC1 in the basolateral plasmalemma of strial marginal and vestibular dark cells confirms conclusions drawn from pharmacological and physiological data. The co-expression of NKCC1 and Na,K-ATPase in highly specialized subpopulations of cochlear and vestibular fibrocytes provides further evidence for their role in recycling K+ leaked or effluxed through hair cells into perilymph back to endolymph, as postulated in current models of inner ear ion homeostasis.
Complex Co-localization of Chromogranins and Neurohormones in the Human Gastrointestinal Tract
Authors: Portela-Gomes Guida Maria , Stridsberg Mats , Johansson Henry , Grimelius Lars
Published: April 2011

Co-localization of chromogranin (Cg) A, B, and C has been studied in different neuroendocrine cell types in histologically normal mucosa from human gastrointestinal tract (corpus, antrum, duodenum, ileum, and colon) using single-, double-, and triple-immunofluorescence stainings. Virtually all enterochromaffin (EC) cells contained CgA, and those in the luminal two thirds of the antral mucosa and villi of small intestine often also contained CgB. A few EC cells in the duodenal crypts contained CgC. Most gastrin cells harbored both CgB and CgA, although rather more CgB than CgA, but some gastrin cells contained all three types, i.e., also CgC. Some CCK cells also contained all three chromogranins. Enteroglucagon cells in the duodenal villi contained CgA and some CgB. CgA (but not B or C) was found in some secretin, GIP, enteroglucagon/peptide YY, and neurotensin cells. A few somatostatin cells contained CgA but neither CgB nor CgC. CgA and C were found mainly in the basal cell region, whereas CgB occurred more diffusely throughout the cytoplasm. This varying distribution suggests that not all secretory granules contain CgA, or that CgB may occur in a nongranular form. The varying composition of the different chromogranins may reflect their complex functional roles in the widespread neuroendocrine system.
PDGFRα Expression During Mouse Embryogenesis: Immunolocalization Analyzed by Whole-mount Immunohistostaining Using the Monoclonal Anti-mouse PDGFRα Antibody APA5
Authors: Takakura Nobuyuki , Yoshida Hisahiro , Ogura Yasunori , Kataoka Hiroshi , Nishikawa Satomi , Nishikawa Shin-Ichi
Published: April 2011

We investigated the cells that express platelet-derived growth factor receptor α (PDGFRα) during mouse embryogenesis. PDGFRα expression has been identified by in situ hybridization or immunohistochemistry using polyclonal antibodies on tissue sections. Because no immunostaining study using whole-mount specimens has been published to date, we established a new monoclonal antibody (MAb), APA5, for this purpose. Our results differed in that APA5 stained only the paraxial mesoderm, whereas other investigators concluded that most if not all mesodermal cells expressed PDGFRα. Moreover, we did not find PDGFRα expression in embryonic erythrocytes, which have been previously suggested to express PDGFRα. On the basis of our present results, we wish to revise the proposed PDGFRα expression as follows. At the pregastrulation stage, PDGFRα is expressed only in primitive endoderm, particularly that in the ectoplacental cone. On gastrulation, it is expressed at high levels in the paraxial mesoderm. This expression continues after its differentiation into the somite. Along with the differentiation and migration of the sclerotome, PDGFRα+ cells begin to become distributed throughout the embryonal mesenchyme. During organogenesis, particularly intense staining is detected in regions of epithelial and mesenchymal interaction, such as the tooth bud and bronchi. In addition to mesodermal derivatives, the developing lens, apical ectodermal ridge, glial precursor, cardiac valves, and choroid plexus express PDGFRα. Our results with whole-mount immunostaining show that PDGFRα is abundantly expressed and may play important roles during embryogenesis.
A Simple Enzyme Histochemical Method for the Simultaneous Demonstration of Acetylcholinesterase and Monoamine Oxidase in Fixed-Frozen Sections
Authors: Dunning Daniel D. , McHaffie John G. , Stein Barry E.
Published: April 2011

We describe an enzyme histochemical technique for the simultaneous demonstration of acetylcholinesterase (AChE) and monoamine oxidase (MAO) (Types A, B, or A+B) in fixed-frozen sections. Several regions in the mesencephalon and brainstem were examined for both somatic and neuropil labeling. The results obtained are equivalent or superior to those obtained using previous methods for the individual localization of these enzymes. The simultaneous visualization of AChE and MAO in the same section allows the relationship of the two enzymes to be easily assessed with brightfield microscopy.
Immunochemical Characterization of a Human Sperm Fibrous Sheath Protein, Its Developmental Expression Pattern, and Morphogenetic Relationships with Actin
Authors: Escalier Denise , Gallo Jean-Marc , Schrével Joseph
Published: April 2011

Among the monoclonal antibodies (MAbs) prepared against human sperm extracts, MAb 4F7 was found to be specific to the human and Macaca fascicularis sperm cytoskeletal fibrous sheath (FS). In Western blotting, MAb 4F7 stains a doublet of polypeptides of about Mr 95 × 103 in extracts of human sperm cells. These polypeptides are not recognized by the KL1 anti-cytokeratin MAb, nor by the MAbs known to bind to the carboxy terminal (IFA) and to the amino terminal (ME101) rod domain of intermediate filaments. Sequential extraction procedures shows that the FS polypeptides recognized by MAb 4F7 are exposed after treatment with 8 M urea. 4F7 immunoreactivity is lost after treatment with high ionic solutions (NaCl, KCl, KI). Immunogold electron microscopy reveals that this protein is present throughout the FS. This FS antigenic determinant first accumulates in an FS proximal body in late spermatids, then in granules extending distally along the flagellum. Staining of spermatozoa with flagellar dysgenesis reveals that this FS protein colocalizes with actin no matter what the location of their abnormal assembly. These data suggest that the transient microtubule-like spindle-shaped body of as yet unknown function could be involved in FS protein deposition and that the assembly of the FS and actin could be under the control of some common morphogenetical factor(s). MAb 4F7 should allow further investigations of this periaxonemal structure in both normal and pathological conditions.
Detection of Intracellular Interleukin-8 in Human Mast Cells: Flow Cytometry as a Guide for Immunoelectron Microscopy
Authors: Grützkau Andreas , Krüger–Krasagakes Sabine , Kögel Heidi , Möller Annelie , Lippert Undine , Henz Beate M.
Published: April 2011

The chemokine interleukin-8 (IL-8) mediates infiltration and adhesion of neutrophils during inflammatory processes. We have previously shown that this cytokine can be produced and released by normal and leukemic human mast cells (HMC-1 cells). To assess whether and to what extent this cytokine is stored intracellularly, we investigated production and localization of IL-8 at the single-cell level by combined use of flow cytometry (FACS) and immunoelectron microscopy. Conditions necessary for optimal fixation and permeabilization of HMC-1 cells were determined by measuring changes in cell-specific light scatter parameters and by estimating cellular uptake of propidiumiodide (PI). In this way, we were able to detect IL-8 with a monoclonal antibody in stimulated cells that were microwave-fixed with a combination of paraformaldehyde (4%) and glutaraldehyde (0.1%), followed by permeabilization with saponin (0.025%). FACS analysis revealed time-dependent synthesis of IL-8 with at most 50% positively stained cells at 8–12 hr after stimulation. For pre-embedding immunogold electron microscopy, cells were treated according to the protocol established by flow cytometry. IL-8 was found to be located in specific cytoplasmic, electron-dense granules of stimulated HMC-1 cells. These results confirm and extend our previous findings by demonstrating IL-8 expression in HMC-1 cells at the single-cell level. In addition, we propose that quantitative FACS can be reliably used in a timesaving manner to establish appropriate conditions for pre-embedding immunoelectron microscopy of intracellular antigens.
Differential Expression of CD66a (BGP), a Cell Adhesion Molecule of the Carcinoembryonic Antigen Family, in Benign, Premalignant, and Malignant Lesions of the Human Mammary Gland
Authors: Riethdorf Lutz , Lisboa Björn W. , Henkel Ute , Naumann Markus , Wagener Christoph , Löning Thomas
Published: April 2011

CD66a, also known as biliary glycoprotein (BGP), is a member of the carcinoembryonic antigen (CEA) family and the human homologue of the rat cell-CAM. There is evidence that aberrant expression or loss of CD66a in tumor tissue is of biological significance. No data about its expression in breast carcinoma cells and only sparse information about the expression of CD66a in normal breast are available thus far. In this study we used monoclonal antibodies to analyze the expression of CD66a and CEA in normal tissue, benign lesions, and in noninvasive and invasive carcinomas of the mammary gland. In normal tissue and benign lesions, CD66a was consistently expressed at the apical sites of epithelial cells and in myoepithelia, whereas CEA was absent or was restricted only to some apical membranes within the ductal tree. The specific staining of myoepithelia was most evident in pseudoinfiltrative radial scars and sclerosing adenosis. However, the apical expression of CD66a disappeared with the development of the malignant phenotype in noninvasive and invasive carcinomas, and changed gradually from low- to high-grade noninvasive carcinomas into a predominant uniform membrane staining all around the atypical cells. CEA expression was irregular in intensity and distribution. The native apical CD66a staining was partially preserved in some highly differentiated invasive carcinomas with a better prognosis, such as tubular and papillary carcinomas. These findings indicate that loss of CD66a expression rather than a change in staining patterns coincides with the development of the malignant phenotype.
Accumulation of Advanced Glycation Endproducts in the Rat Nephron: Link with Circulating AGEs During Aging
Authors: Verbeke Philippe , Perichon Martine , Borot–Laloi Caroline , Schaeverbeke Jean , Bakala Hilaire
Published: April 2011

The accumulation of advanced glycosylation end products (AGEs) is believed to be a factor in the development of aging nephropathy. We have attempted to establish a link between the formation of AGEs and the onset of renal impairment with aging, indicated by albuminuria, using a fluorescence assay and immunohistochemical detection of AGEs in the renal extracellular matrix in rats. The fluorescence of collagenase-digested Type IV collagen from GBM increased with age, from 1.65 ± 0.05 AU/mM OHPro (3 months) and 1.58 ± 0.04 (10 months) to 2.16 ± 0.06 (26 months) ( p>0.001) and 2.53 ± 0.18 (30 months) ( p>0.001). In contrast, the extent of early glycation products significantly decreased from 5.35 ± 0.25 nmol HCHO/nmol OHPro at 3 months to 3.14 ± 0.19 at 10 months ( p>0.001), 3.42 ± 0.38 at 26 months, and 0.74 ± 0.08 at 30 months ( p>0.001). The urinary fluorescence of circulating AGE rose from 2.42 ± 0.15 AU/mg protein (3 months), 1.69 ± 0.07 (10 months), to 4.63 ± 0.35 (26 months) ( p>0.01) and 4.73 ± 0.72 (30 months), while the serum fluorescence increased from 0.39 ± 0.02 AU/mg protein at 3 months and 0.43 ± 0.02 at 10 months to 0.59 ± 0.04 at 26 months ( p>0.001) and 0.54 ± 0.03 at 30 months ( p>0.04). Polyclonal antibodies raised against AGE RNase showed faint areas of AGE immunoreactivity in mesangial areas in the nephrons of young rats. The immunolabeling of Bowman's capsule, the mesangial matrices, and the peripheral loops of glomerular and tubule basement membranes increased with rat age. The increase in circulating AGE peptides parallels the accumulation of AGEs in the nephron, and this parallels the pattern of extracellular matrix deposition, suggesting a close link between AGE accumulation and renal impairment in aging rats. (J Histochem Cytochem 45:1059–1068, 1997)
Cytochemical Localization of Ouabain-sensitive, K+-dependent p-nitrophenylphosphatase Activity in the Facial Nerve of Reserpinized Guinea Pigs
Authors: Kanoh Naoyuki
Published: April 2011

Ion-transporting Na,K-ATPase plays an essential role in nerve conduction. To clarify the cytochemical effects of reserpine on transport Na,K-ATPase activity, the localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K-NPPase) activity was investigated in the facial nerves of normal and reserpinized guinea pigs using a cerium-based method. In the normal facial nerve, the reaction product of K-NPPase activity was observed on the internodal axolemma and Schmidt-Lanterman incisures. In the Ranvier nodes, enzyme activity was localized to the paranodal and nodal axolemma. In the reserpinized nerves, reaction product was detectable on the nodal axolemma but was undetectable on the other parts of the axolemma. Nodal K-NPPase was not affected by reserpine treatment. Therefore, the transport Na,K-ATPase on the nodal axolemma might differ from that on the other parts of the axolemma. Allowing reserpinized animals to survive. Two different ouabain-sensitive K-NPPase reactivities, “reserpine-sensitive” and “reserpine-resistant,” might be present in the facial nerve of guinea pigs. (J Histochem Cytochem 45:1129–1135, 1997)
Immunohistochemical Analysis of DNA Synthesis During Chronic Stimulation with Isoproterenol in Mouse Submandibular Gland
Authors: Matsuura Sachiko , Suzuki Kazuo
Published: April 2011

We investigated the numbers of DNA-synthesizing cells in mouse submandibular glands (SMGs) during chronic isoproterenol (IPR) administration, using an immunohistochemical method with anti-bromodeoxyuridine (BrdU) antibodies. Adult and immature female mice were injected with IPR daily or every other day up to a total of seven injections. Whereas hypertrophic enlargement of acinar cells continued with repeated IPR injections, induction of DNA synthesis was transient. With one IPR injection, more than 50% of the acinar cells were induced to synthesize DNA, but after five daily injections the numbers of cells in the DNA synthetic phase decreased and returned to control values. This pattern was basically the same for immature mice and for adult mice stimulated by IPR every other day. The responsiveness of the SMGs of immature mice to IPR was somewhat lower than that of adults. Prolongation of the interval between injections of IPR, compared to daily injections, did not cause sustained induction of DNA synthesis. These data suggest that the hypertrophic enlargement and the induction of DNA synthesis by IPR are mediated through different intracellular pathways after the stimulation of β-adrenergic receptors, and that the regulatory mechanism for the induction of DNA synthesis is complex. (J Histochem Cytochem 45:1137–1145, 1997)
Visualization of β-Galactosidase by Enzyme and Immunohistochemistry in the Olfactory Bulb of Transgenic Mice Carrying the LacZ Transgene
Authors: Sekerková Gabriela , Katarova Zoya , Joó Ferenc , Wolff Joachim R. , Prodan Simona , Szabó Gábor
Published: April 2011

In the olfactory bulb (OB) of a transgenic mouse line that carries the bacterial LacZ gene under the control of the 5'-regulatory region of the GAD67 gene, expression of the β-galactosidase was confined almost exclusively to the non-GABAergic mitral and tufted cells. By light microscopy, enzyme histochemistry showed strong staining in the cell bodies and faint diffuse staining in the axons and dendrites. With immunohistochemistry for β-galactosidase the entire cytoplasm, including the axons and dendrites, was strongly stained. By electron microscopy, β-galactosidase enzyme histochemistry resulted in a submicroscopic reaction product that was diffusely distributed in the cytoplasm of neurons. In addition, large deposits of the reaction product were also seen attached to the cytoplasmic side of the membranes. In contrast, when the intracellular localization of β-galactosidase was determined by immunohistochemistry, homogeneous cytoplasmic staining was obtained that filled the entire cytoplasm including the terminal dendrites and fine axons. Therefore, synaptic contacts of the β-galactosidase-positive output neurons with other β-galactosidase-negative neuronal cells were readily recognized in the OB. As we demonstrated, transgenic mouse lines expressing the LacZ reporter gene in a well-defined neuronal subpopulation can be used to follow β-galactosidase-positive neurons and to directly identify their synaptic connections. (J Histochem Cytochem 45:1147–1155, 1997)
Patterns of Hyaluronan Staining Are Modified by Fixation Techniques
Authors: Lin Weiqi , Shuster Svetlana , Maibach Howard I. , Stern Robert
Published: April 2011

The apparent intensity of hyaluronan (HA) staining in tissue sections can vary as a function of fixation techniques. We examined the histochemical distribution of HA in normal human skin using an HA-specific binding peptide derived from bovine nasal cartilage. The HA, particularly in the dermis, was best preserved in sections fixed in 10% acid-formalin with 70% ethanol. In contrast, sections fixed in the routine 10% neutral-buffered formalin had a much weaker intensity of HA staining. Furthermore, acid-formalin/ethanol-fixed sections retained much of their apparent HA after incubation with saline, in contrast to the neutral formalin-fixed sections, in which most of the stainable HA was lost. Such marked differences in staining intensity were not observed in slides stained with Alcian blue, a procedure pressumed to stain HA as well as other glycosaminoglycans. Staining using the HA binding peptide was entirely absent when sections were first preincubated in hyaluronidase, whereas similar Alcian blue-stained sections retained most of their staining intensity. Caution should be exercised in evaluating the distribution of HA in tissues using the HA binding peptide, particularly when different fixation techniques among several laboratories are being compared. In addition, the ability to evaluate the HA content of tissues using Alcian blue staining should be reconsidered. The sulfated glycosaminolglycans of the “ground substance” appear to be the predominant substrates for Alcian blue.

(J Histochem Cytochem 45:1157–1163, 1997)
Localization of BRCA1 Gene Expression in Adult Cynomolgus Monkey Tissues
Authors: Durocher Francine , Simard Jacques , Ouellette Johanne , Richard Virgile , Labrie Fernand , Pelletier Georges
Published: April 2011

The breast and ovarian cancer susceptibility gene BRCA1 encodes a phosphoprotein of 1863 amino acids containing a highly conserved N-terminal RING finger domain and a C-terminal acidic region typical of several transcription factors. BRCA1 acts as a tumor suppressor that may inhibit the proliferation of breast and ovarian cancer cells. To gain knowledge and to further understand the biological function of BRCA1, we examined its localization and expression in various tissues from 20-year-old male and female cynomolgus monkeys ( Macaca fascicularis) by in situ hybridization using a 35S-labeled human BRCA1 DNA probe fragment derived from exon 11. In mammary glands, BRCA1 expression was primarily located in the duct and acinar epithelial cells. In the ovary, strong BRCA1 expression was detected in granulosa cells in maturing follicles and in luteal cells of the corpus luteum, as well as in the epithelial cells overlying the tunica albuginea. Specific signal was also observed in epithelial cells of the oviduct, endometrium, cervix, and vagina. Moreover, BRCA1 was strongly expressed in the germinal epithelium of the seminiferous tubules as well as over interstitial cells of the testis, in the epithelium of the epididymis, and in epithelial cells bordering the glandular lumen of the seminal vesicles. Signal was also detected in both the anterior and posterior lobes of the pituitary. In the adrenal glands, the signal was greater in the zona glomerulosa compared to the two other cortical zones, whereas the medullary cells were weakly labeled. In the stomach, and in small and large intestine, epithelial cells of the crypts usually exhibited stronger positive reaction than that observed over surface epithelial lining cells. BRCA1 expression was also found in diverse types of epithelial cells of the thyroid, pancreas, salivary glands, trachea, urinary bladder, and kidneys. In addition to demonstrating widespread tissue- and cell-specific expression of the BRCA1 gene in primate tissues, primarily in the epithelia, we observed a weaker but specific signal in various other cell types, suggesting a generalized biological function of BRCA1.
Crosslinked Plasmalemmal Cholesterol Is Sequestered to Caveolae: Analysis with a New Cytochemical Probe
Authors: Fujimoto Toyoshi , Hayashi Masami , Iwamoto Machiko , Ohno-Iwashita Yoshiko
Published: April 2011

Θ-Toxin (perfringolysin O), a cholesterol-binding toxin, was partially proteolyzed and biotinylated (BCΘ) to eliminate hemolyzing activity and was used as a cytochemical probe. In fixed cells, binding of BCΘ was intense in the plasma membrane, especially at the base of apical microvilli and in lateral processes. The labeling was abolished by pretreatment with filipin, digitonin, or tomatin. When living cultured cells were treated with BCΘ and then with either fluorescein–avidin D or colloidal gold–streptavidin, the labeling in fine dots was distributed on the cell surface without local concentration as long as cells were kept on ice. When the temperature was raised to 37C after treatment, the probe formed discrete large patches and became sequestered to caveolae. Binding of BCΘ alone without the secondary reagents did not cause redistribution even at 37C. Because the plasma membrane maintains integrity even after binding of BCΘ, the probe can be used not only for cytochemical labeling of fixed cells but for pursuing the behavior of crosslinked cholesterol molecules in living cells. By use of this new probe, the present study revealed that crosslinked cholesterol in the plasma membrane is sequestered to caveolae.
Peptide Expression in GABAergic Neurons in Rat Suprachiasmatic Nucleus in Comparison with Other Forebrain Structures: A Double Labeling In Situ Hybridization Study
Authors: Tanaka Masaki , Matsuda Tomoyuki , Shigeyoshi Yasufumi , Ibata Yasuhiko , Okamura Hitoshi
Published: April 2011

We investigated the characteristics of GABAergic neurons in the rat suprachiasmatic nucleus (SCN) in normal untreated rats by examination of co-expressed peptides. We adopted double labeling in situ hybridization using a digoxigenin-labeled glutamic acid decarboxylase (GAD) riboprobe and 35S-labeled peptide riboprobes. GAD mRNA-positive neurons were distributed throughout the SCN from the rostral to the caudal pole. In the dorsomedial part of the SCN, most GAD mRNA-positive neurons co-expressed arginine vasopressin mRNA. In the ventrolateral part of the SCN, about two thirds of GAD mRNA-positive neurons co-expressed vasoactive intestinal peptide (VIP) mRNA. Co-expression of GAD and somatostatin mRNA was observed in virtually all neurons of the intermediate part of the SCN. In contrast, these peptidergic traits were poorly expressed in hypothalamic GABAergic neurons outside the SCN. Vasopressin mRNA-positive cells in the supraoptic nucleus did not express GAD mRNA, and co-expression of somatostatin mRNA and GAD mRNA was rare in the periventricular hypothalamic nucleus. Similarly, the VIP mRNA co-expression ratio of GABAergic neurons in the cerebral cortex was far lower than that in the SCN.
Platinum Porphyrins as Phosphorescent Label for Time-resolved Microscopy
Authors: de Haas Richard R. , van Gijlswijk Rob P.M. , van der Tol Erik B. , Zijlmans Henry J.M.A.A. , Bakker–Schut Tom , Bonnet Jan , Verwoerd Nico P. , Tanke Hans J.
Published: April 2011

We investigated phosphorescent metalloporphyrins as potential labels for time-resolved microscopy. On the basis of spectroscopic analysis of their physicochemical properties (quantum yield, molar absorption coefficient, decay times) the best candidates were selected. Next, we synthesized antibody and avidin metalloporphyrin conjugates. The optimal F/P ratio with respect to quantum yield, decay time, and retention of biological activity of these immunoreagents was determined. The reagents were then evaluated by in situ hybridization and immunocytochemical procedures for demonstration of hapten-labeled DNA probes, membrane antigens (CD type), and 28S rRNA. All stained samples exhibited bright phosphorescence that could be selectively detected using time-resolved microscopy, especially when glucose/glucose oxidase was added to the embedding medium to deplete oxygen. Applications of time-resolved detection of phosphorescent porphyrins in strongly autofluorescent material (histological sections) are discussed.
Automated Double Labeling of Proliferation and Apoptosis in Glutathione S-transferase-positive Hepatocytes in Rats
Authors: Short Brian G. , Zimmerman Dawn M. , Schwartz Lester W.
Published: April 2011

Immunohistochemical markers for proliferation (bromodeoxyuridine, BrdU) and apoptosis (in situ terminal deoxynucleotide transferase dUTP nick end-labeling, TUNEL) were localized within glutathione S-transferase (GSTP)-positive hepatic foci in rats. Using the TechMate Automated Staining System (BioTek Solutions; Santa Barbara, CA), formalin-fixed, paraffin-embedded sections were run through a double-label avidin–biotin–immunoperoxidase protocol in less than 10 hr. Steam heat-induced epitope retrieval and/or proteolytic digestion preceded each labeling procedure. Color development was achieved using diaminobenzidine (DAB) with nickel enhancement for BrdU and TUNEL and VIP for GSTP. Results illustrate clear staining, brown-black BrdU-positive nuclei or TUNEL-positive apoptotic bodies within purple GSTP-positive hepatocytes. This automated procedure provides a method to easily identify and quantitate proliferating or apoptotic cells within foci of altered hepatocytes in rat liver and may have general applications for studies of cell or tissue kinetics during development, differentiation, and various pathological conditions in animals and humans.
Detection of the Receptor for the Human Urokinase-type Plasminogen Activator Using Fluoresceinated uPA
Authors: Ciccocioppo Rosalba , Capri Maria G. , Alberti Saverio
Published: April 2011

The urokinase-type plasminogen activator (uPA) is a serine protease that plays a crucial role in blood coagulation and in tumor invasion and metastasis. uPA is a relatively large polypeptide and binds the uPA receptor (uPAR) with high affinity and specificity. Therefore, it was a good candidate for direct labeling with a fluorochrome for detection of the uPAR. We have produced a fluorescein (FITC)-labeled human uPA using a conjugation procedure that did not significantly alter its binding characteristics to the uPAR. Thirty nM FITC–uPA efficiently stains 2 × 105 uPAR-transfected mouse cells in suspension, as determined by flow cytometric analysis. One μg of FITC–uPA efficiently stains 2 × 105 uPAR transfectants grown on slides and analyzed by fluorescence optical microscopy. Human cell lines expressing the endogenous uPAR were stained with similar efficiency. Fixation in paraformaldehyde only slightly reduced the efficiency of staining of both transfectants and cell lines. These characteristics allow the use of FITC–uPA in both static and dynamic morphological studies of uPAR-expressing cells.
Different Concentrations of Mg++ Ions Affect Nuclear Matrix Protein Distribution During Thermal Stabilization of Isolated Nuclei
Authors: Neri Luca M. , Capitani S. , Valmori Aurelio , Riederer Beat M. , Martelli Alberto M.
Published: April 2011

The nuclear matrix, a proteinaceous network believed to be a scaffolding structure determining higher-order organization of chromatin, is usually prepared from intact nuclei by a series of extraction steps. In most cell types investigated the nuclear matrix does not spontaneously resist these treatments but must be stabilized before the application of extracting agents. Incubation of isolated nuclei at 37C or 42C in buffers containing Mg++ has been widely employed as stabilizing agent. We have previously demonstrated that heat treatment induces changes in the distribution of three nuclear scaffold proteins in nuclei prepared in the absence of Mg++ ions. We studied whether different concentrations of Mg++ (2.0–5 mM) affect the spatial distribution of nuclear matrix proteins in nuclei isolated from K562 erythroleukemia cells and stabilized by heat at either 37C or 42C. Five proteins were studied, two of which were RNA metabolism-related proteins (a 105-kD component of splicing complexes and an RNP component), one a 126-kD constituent of a class of nuclear bodies, and two were components of the inner matrix network. The localization of proteins was determined by immunofluorescent staining and confocal scanning laser microscope. Mg++ induced significant changes of antigen distribution even at the lowest concentration employed, and these modifications were enhanced in parallel with increase in the concentration of the divalent cation. The different sensitivity to heat stabilization and Mg++ of these nuclear proteins might reflect a different degree of association with the nuclear scaffold and can be closely related to their functional or structural role.
Immunocytochemical Localization of Chymase to Cytoplasmic Vesicles After Rat Peritoneal Mast Cell Stimulation by Compound 48/80
Authors: Login Gary R. , Dvorak Ann M. , Yamakawa Midori , Digenis Eleni C. , Tanda Naoko , Aoki Mikako , Lunardi Laurelúcia O. , Schwartz Lawrence B.
Published: April 2011

The subcellular events responsible for release of mediators by mast cells may help to clarify roles for mast cells in health and disease. In this study we show that the granule-associated protease chymase is also within cytoplasmic vesicles in appropriately stimulated rat peritoneal mast cells. Rat peritoneal mast cells were recovered before or 1–10 sec after exposure to the secretogogue compound 48/80 (10 μg/ml) and then were examined by radioimmunoassay to quantify histamine release or were processed, using routine methods for postembedding immunoelectron microscopy, to identify the subcellular localization of chymase. In comparison to unstimulated cells, compound 48/80 stimulated cells in two independent experiments showed an increase (15%, 28%) in the surface area of the cell and a decrease (12%, 6%) in the surface area of the total granule compartment before degranulation channel formation. These global cellular changes occurred in a background of transient but significant ( p<0.01) increases in the area and number of chymase-immunoreactive vesicles per μ2 cytoplasm. These changes were detectable at 5 or 7 sec after stimulation with compound 48/80 but returned to near prestimulation levels by 9 or 10 sec after addition of compound 48/80 (total cumulative histamine release was 28% by 8 sec and 47% by 14 sec). These observations suggest that vesicles participate in the early stages of regulated secretion of chymase from rat peritoneal mast cells.
Quantitative Computerized Image Analysis of Tn and T (Thomsen–Friedenreich) Epitopes in Prognostication of Human Breast Carcinoma
Authors: Wang Bao-Le , Springer Georg F. , Carlstedt Sheila C.
Published: April 2011

The precursors of the blood group N and M-immunodominant structures, Tn and T (Thomsen–Friedenreich) epitopes (EPs) occur in ∼90% of carcinomas (CAs) but are masked in benign-diseased and healthy tissues. We determined quantitatively on 55 primary invasive ductal breast CAs, stages I to IV, the prognostic value of extent of Tn and T EP expression over an observation period exceeding 5 years postoperatively. Classical, established pathological and histological prognostic characteristic indicators associated with survival were subdivided by standard criteria into favorable and unfavorable categories. Tissue sections were reacted with monoclonal anti-Tn and -T antibodies, followed by the streptavidin–biotin–peroxidase–DAB procedure; counterstain was methyl green. Tn and T EPs were then quantitated by computerized image analysis. Of the 55 CAs, 51 clearly expressed Tn and T, and four had traces. Strong Tn EP expression was statistically significantly associated with shortened 5-year disease-free interval, increasing pTNM stages, positive lymph node status, and increasing combined histological grades. T EPs were usually well expressed but showed no significant association with prognostic factors. Our results suggest that quantitative immunohistochemistry–image analysis of Tn EPs of primary breast CAs may add new parameters to prognostication.
Effects of Tissue Protectants on the Kinetics of Lactate Dehydrogenase in Cells
Authors: Nakae Yoshiko , Stoward Peter J.
Published: April 2011

We studied the effects of two tissue protectants, polyvinyl alcohol (PVA) and agarose gel, on a kinetic parameter of lactate dehydrogenase LDH that is assumed to be related to the extent of diffusion of the enzyme out of tissue sections during its histochemical assay. The kinetics of the enzyme in mouse gastrocnemius (skeletal) muscle fibers and periportal hepatocytes were determined in unfixed sections incubated either on substrate (l-lactate)-containing agarose gel films or in aqueous assay media in the presence or absence of 18% PVA. The absorbances of the formazan final reaction products at their isobestic point were measured continuously in the cytoplasm of individual cells as a function of incubation time, using a real-time image analysis system. Whichever incubation medium was used, the absorbances in the two cell types increased nonlinearly during the first minute of incubation but linearly for incubation times between 1 and 3 min. The nonlinearity of the LDH reaction was analyzed using the equation vi - v = a° A, where vi is the observed initial velocity determined from the absorbance changes during the first 10 sec of incubation and v and ° A are respectively the gradient and intercept on the absorbance axis of the linear regression line of the absorbance on incubation times between 1 and 3 min. The plots of the observed ( vi - v) against ° A were linear. Their gradients a were characteristic for each cell type and tissue protectant. The a values for skeletal muscle fibers were 12–43% lower than those for hepatocytes. The a value for hepatocytes obtained with the PVA method was 32% lower than that determined with the gel film method. For skeletal muscle fibers, the a values determined by the two methods were almost the same. Addition of excess pyruvate to the aqueous assay medium had no effects on a for either muscle fibers or hepatocytes. In contrast, a was zero for sections of polyacrylamide gels containing purified enzyme, whether incubated on agarose films or in PVA media. These data confirmed that the constant a is related to the extent to which the enzyme diffuses out of sections during incubation but not to product inhibition of LDH by pyruvate. PVA was more effective for protecting diffusion of LDH from hepatocytes than from skeletal muscle fibers, possibly because hepatocytes contain a greater proportion of diffusable (unbound) LDH than skeletal muscle fibers.
Differential Localization of Superoxide Dismutase Isoforms in Placental Villous Tissue of Normotensive, Pre-eclamptic, and Intrauterine Growth-restricted Pregnancies
Authors: Myatt Leslie , Eis Annie L.W. , Brockman Diane E. , Kossenjans Wilhelm , Greer Ian A. , Lyall Fiona
Published: April 2011

Several isoforms of superoxide dismutase (SOD), including copper/zinc (cytosolic) and manganese (mitochondrial), exist. In the human placenta, SOD may prevent excessive superoxide accumulation and any potential deleterious oxidative effects. In pre-eclampsia, increased levels of lipid peroxide and decreased SOD activity have been described in the placenta. Oxidative stress such as occurs in pre-eclampsia can alter expression of SOD isoforms. The objective of this study was to localize the copper/zinc and manganese SOD isoforms in the placenta using immunohistochemistry and to compare localization and intensity of immunostaining in tissues from normotensive pregnancies with those from pregnancies complicated by pre-eclampsia and/or intrauterine growth restriction (IUGR). Western blotting with specific antibodies recognized a 17-kD copper/zinc and a 23-kD manganese SOD subunit in placental homogenates. Intense immunostaining for the manganese SOD isoform was seen in villous vascular endothelium, but only faint staining was found in the syncytiotrophoblast or villous stroma. In serial sections, intense immunostaining for copper/zinc SOD was seen in certain cells of the villous stroma but only faint immunostaining in syncytiotrophoblast and vascular endothelium. No apparent differences in localization or intensity of immunostaining for either isoform were seen between tissues of normotensive or pre-eclamptic pregnancies, with or without IUGR. The different cellular localizations of the SOD isoforms suggest that they fulfill different functional roles within the placenta.