bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Epidermal Growth Factor Receptor in Fetal Mouse Submandibular Gland Detected by a Biotinyltyramide-based Catalyzed Signal Amplification Method
Authors: Gresik Edward W. , Kashimata Masanori , Kadoya Yuichi , Mathews Robin , Minami Naomi , Yamashina Shohei
Published: April 2011

Branching morphogenesis of the fetal mouse submandibular gland (SMG) can be modulated in vitro by stimulation or inhibition of the epidermal growth factor receptor (EGFR). Because the mRNAs for EGF and EGFR are detectable in RNA of SMG rudiments isolated directly from fetuses, the EGF system probably operates physiologically as a regulator of SMG morphogenesis. However, neither EGFR protein nor its precise cellular localization has been characterized in the fetal SMG. Here we show EGFR protein in fetal mouse SMG by immunoprecipitation, affinity labeling, ligandinduced autophosphorylation, and immunohistochemistry. SMGs from E16 fetuses (day of vaginal plug = EO) were labeled with [35S]-cysteine/methionine and homogenized. After addition of specific antibody to EGFR, the immunoprecipitate was isolated, resolved by polyacrylamide gel electrophoresis, and detected by autoradiography. A single band of 170 kD was detected, corresponding to the EGFR protein. Affinity labeling with [125I]-EGF of the membrane fraction of E18 SMG also revealed a prominent band at 170 kD, showing that this EGFR protein can bind specifically to its ligand. Incubation of SMG membranes from E18 fetuses with EGF in the presence of [γ-32P]-ATP, followed by immunoprecipitation with anti-phosphotyrosine antibody also showed a single band at 170 kD, demonstrating autophosphorylation of the EGFR in response to binding of its ligand. Immunohistochemical localization of the cellular sites of EGFR in the fetal SMG required use of a catalyzed signal amplification procedure, with biotinyltyramide as the amplifying agent. EGFR was localized predominantly, if not exclusively, in cell membranes of epithelial cells of the rudiment, whereas staining of mesenchymal cells was equivocal. Staining was strongest on duct cells, and weak on cells of the end-pieces. These findings clearly show that a functional EGFR protein is expressed in fetal SMG chiefly, if not exclusively, on epithelial cells.
Constant Detection of CD2, CD3, CD4, and CD5 in Fixed and Paraffin-embedded Tissue Using the Peroxidase-mediated Deposition of Biotin-Tyramide
Authors: Malisius Rainer , Merz Hartmut , Heinz Boris , Gafumbegete Evariste , Koch Britta U. , Feller Alfred C.
Published: April 2011

Immunohistochemical methods are widely used for diagnostic purposes in histopathology. However, the use of most monoclonal anti-leukocyte antibodies is limited to frozen tissues. Initially, it was believed that formalin fixation in particular, which is the gold standard for morphological tissue preservation, destroys most of the antigen binding sites. In recent years, protease digestion and the introduction of microwave techniques have significantly enhanced the sensitivity of immunohistochemical techniques, and a variety of hidden antigen sites in formalin-fixed tissue have been retrieved for initially unreactive antibodies. It therefore became clear that many of the leukocyte antigens are not irreversibly destroyed but are most probably masked during the fixation process. We developed a technique combining optimized pretreatment of formalin-fixed tissue with a dramatic enhancement of the immunohistochemical sensitivity and named it the ImmunoMax method. The ImmunoMax method proves that by optimizing the technique at the following three levels it is possible to detect formalin-sensitive leukocyte antigens: (a) standard fixation of the tissue; (b) sufficient antigen unmasking; and (c) increasing the substrate turnover by multiplication of binding sites with subsequent enhancement of the immunohistochemical reaction. Using this optimized ImmunoMax method, we were able to detect CD2, CD3, CD4, and CD5 with conventional monoclonal antibodies in formalin-fixed, paraffin-embedded tissue specimens of various lymphoid tissues.
Freeze-fracture Cytochemistry: A New Method Combining Immunocytochemistry and Enzyme Cytochemistry on Replicas
Authors: Takizawa Toshihiro , Saito Takuma , Robinson John M.
Published: April 2011

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphatase activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.
p53 Expression in Human Carcinomas: Could Flow Cytometry Be an Alternative to Immunohistochemistry?
Authors: Benini Elvira , Costa Aurora , Abolafio Gabriella , Silvestrini Rosella
Published: April 2011

Several studies have shown that p53 ression has important clinical implications as an indicator of prognosis and response to chemotherapy or radiotherapy in different human tumor types. Determination of p53 expression by immunohistochemistry (IHC) has been incorporated into routine practice and its reliability has been consolidated. However, flow cytometric (FCM) analysis might represent an important objective and rapid approach. In the present study we determined p53 expression by IHC and FCM on a series of 118 human solid tumors. IHC determination was performed on histological sections and FCM analysis on cell suspensions. Low correlation coefficients (r5 from 0.22 to 0.57) were observed between IHC and FCM data from individual tumors. By considering the IHC approach as the gold standard, high sensitivity and low specificity were found for FCM in detecting p53 expression. The FCM analysis of p53 expression and DNA content showed p53-positive cells in all cell cycle phases. Moreover, in most breast, lung, and colon aneuploid tumors (77%), p53-positive cells were detected only in the subpopulations with abnormal DNA content. In conclusion, FCM-p53 expression cannot be used alternatively to IHC determination, and its clinical relevance remains to be validated. Nevertheless, FCM may provide important information about p53 protein expression in the different subpopulations and cell cycle phases.
Effects of C-Mpl Ligand on Cytoplasmic Maturation of Murine Megakaryocytes and on Platelet Production
Authors: Ishida Yoji , Ito Toshiharu , Kuriya Shin-ichiro
Published: April 2011

To test the hypothesis that the c-mpl ligand is not a primary factor in thromb-ocytopoiesis, we investigated the biological effects of recombinant human (rh) c-mpl ligand on differentiation of murine progenitor cells and on maturation of the cultured murine megakaryocytes under serum-free conditions on the basis of ploidy distribution, megakaryocyte/platelet-specific surface antigen CD 61 [glycoprotein (GP) IIIa], and cytoplasmic acetylcholinesterase (AchE) expression in vitro. In addition, we studied the effect of c-mpl ligand on proplatelet formation (PPF) by murine mature megakaryocytes. AchE was less strongly expressed in cultured megakaryocytic cells stimulated by c-mpl ligand than in those stimulated by recombinant murine (rm) IL-3 + rh IL-6 during the differentiation of progenitor cells. Less CD 61 was expressed by c-mpl ligand during both the differentiation of progenitor cells and the maturation of megakaryocytes compared with that by rm IL-3 + rh IL-6. Endomitosis, however, was more stimulated by c-mpl ligand than by rm IL-3 + rh IL-6 under both conditions. Furthermore, PPF of mature megakaryocytes was not stimulated by c-mpl ligand. These results indicate that c-mpl ligand stimulates the nuclear development of megakaryocytic cells but that it does not stimulate cytoplasmic maturation and PPF as much as IL-6. These data strongly suggest that c-mpl ligand is not a primary factor in platelet production.
Expression of Prostaglandin G/H Synthase Type 1, but not Type 2, in Human Ovarian Adenocarcinomas
Authors: Doré Monique , Cóté Lucie C. , Mitchell Andrew , Sirois Jean
Published: April 2011

Prostaglandin endoperoxide synthase (PGHS) is a key rate-limiting enzyme in prostaglandin biosynthesis. PGHS has recently been shown to be expressed in human colorectal cancers and in experimental cutaneous papillomas and carcinomas. However, PGHS expression has not been investigated in ovarian cancers. The objectives of this study were to determine whether PGHS isoenzymes are expressed in human ovarian cancer and, if so, to identify which isoform is involved (PGHS-1 and/or PGHS-2) and to characterize its cellular localization. Sixteen human ovarian adenocarcinomas were studied by immunohistochemistry using specific antibodies recognizing PGHS-1 or PGHS-2. Results showed that all adenocarcinomas demonstrated the presence of tumor cells expressing PGHS-1 but not PGHS-2. Patterns of staining of tumor cells varied among different types of adenocarcinomas, with cells presenting either a mostly diffuse cytoplasmic immunoreactivity or, alternatively, a staining mainly concentrated around the nucleus. No correlation between the intensity of the immunostaining and the degree of malignancy of tumors could be established ( r −0.03: p>0.05). Immunoblot analysis with PGHS-1-selective antibodies of cell extracts from adenocarcinomas revealed the presence of a characteristic 72,000 Mr immunoreactive band. Therefore, these results show for the first time that PGHS-1 is expressed in human ovarian adenocarcinomas.
Immunolocalization of Vacuolar-type H+-ATPase in Rat Submandibular Gland and Adaptive Changes Induced by Acid–Base Disturbances
Authors: Roussa Eleni , Thévenod Frank , Sabolic Ivan , Herak–Kramberger Carol M. , Nastainczyk Wolfgang , Bock Rudolf , Schulz Irene
Published: April 2011

Using antibodies against the 31-kD and 70-kD subunits of vacuolar type H+-ATPase (V-ATPase) and light microscopic immunocytochemistry, we have demonstrated the presence of this V-ATPase in rat submandibular gland. We have also investigated the adaptive changes of this transporter during acid-base disturbances such as acute and chronic metabolic acidosis or alkalosis. Our results show intracellularly distributed V-ATPase in striated, granular, and main excretory duct cells in controls, but no V-ATPase immunoreaction in acinar cells. Both acute and chronic metabolic acidosis caused a shift in V-ATPase away from diffuse distribution towards apical localization in striated and granular duct cells, suggesting that a V-ATPase could be involved in the regulation of acid–base homeostasis. In contrast, during acidosis the main excretory duct cells showed no changes in the V-ATPase distribution compared to controls. With acute and chronic metabolic alkalosis, no changes in the V-ATPase distribution occurred.
Immunocytochemical Localization of the Prohormone Convertases PC1 and PC2 in Rat Prolactin Cells
Authors: Muller Laurent , Picart Renée , Barret Alain , Seidah Nabil G. , Tougard Claude
Published: April 2011

The prohormone convertases PC1 and PC2 are subtilisin-related endopepti-dases that process prohormone and neuropeptide precursors. Using different ultrastructural immunocytochemical approaches, we have investigated their intracellular distribution in a neuroendocrine cell type that has not been examined thus far, the rat anterior pituitary lactotrope. These cells secrete mainly prolactin and also express the neuroendocrine-specific protein secretogranin II, which is considered a peptide precursor. Our study provides evidence for the expression of PC1 and PC2 in rat lactotropes and provides new information on their subcellular localization. Apart from their presence in the secretory granules, PC1 and PC2 displayed different major localization along the secretory pathway. PC1 immunoreactivity was concentrated in the Golgi apparatus, whereas PC2 immunoreactivity was prominent in the rough endoplasmic reticulum (RER). These observations provide morphological support for previous biochemical analysis of proPC1 and proPC2 post-translational processing, which has demonstrated that PC1 exits very rapidly from the RER, whereas PC2 is retained much longer in this compartment.
Integrin Expression in Developing Smooth Muscle Cells
Authors: Wang Rong , Stromer Marvin H. , Huiatt Ted W.
Published: April 2011

We studied the specific expression patterns and distributions of α1 and β1 integrin subunits, the major cell adhesion receptors in smooth muscle, in developing smooth muscle cells from 16-, 18-, and 20-day embryonic gizzards and from 1- and 7-day post hatch chick gizzards by SDS-PAGE, immunoblotting, and immunoelectron microscopy. Antibodies raised against α1 and β1 integrins isolated from avian gizzards were used as probes. Gels and blots showed that the amount of α1 and β1 integrins increased as age increased, with major increases at 1 and 7 days post hatch. Image analysis of immunoelectron micrographs demonstrated that statistically significant labeling increases occurred between embryonic Days 16 and 18, between embryonic Day 20 and 1 day post hatch, and between 1 day and 7 days post hatch. Immunolabeling with both anti-α1 and anti-β1 integrin was prominent at membrane-associated dense plaques (MADPs) and at filament anchoring regions at cell ends. This indicates that α1 and β1 integrin expression coincides temporally with the intracellular proliferation and reorientation of myofilaments. The similarity in distribution patterns of α1 and β1 integrins during development suggests that the two integrin subunits are synchronously expressed during development and do not appear sequentially.
Use of Methacrylate De-embedding Protocols for In Situ Hybridization on Semithin Plastic Sections with Multiple Detection Strategies
Authors: Warren Kelley C. , Coyne Kathryn J. , Waite J. Herbert , Cary S. Craig
Published: April 2011

New plastic resins are gradually replacing traditional paraffin-embedding in situ hybridization (ISH) strategies. One unique resin that has not been fully investigated or exploited with respect to light microscopic ISH is a methacrylate mixture. The advantage of this resin is its ability to be removed from tissues postsectioning, dramatically increasing hybridization signal compared to that obtained in other plastics. The goal of this study was to evaluate the general applicability of the methacrylate embedding acetone de-embedding (MEADE) technique for ISH investigations. Several high-resolution, high-sensitivity ISH protocols are described, using both end-labeled oligonucleotides and randomly primed DNA probes (200–400 bps), signal amplification by catalyzed reporter deposition (CARD), and chromogenic and fluorescent detection methods. With slight modification, the MEADE ISH technique permitted localization of bacterial symbionts in fragile gill tissue and collagen transcripts in foot tissue of two marine bivalves. MEADE ISH has proved extremely versatile and will likely be suitable for many future applications.
Characterization of Protease-activated Receptor-2 Immunoreactivity in Normal Human Tissues
Authors: R. D'Andrea Michael , Derian Claudia K. , Leturcq Didier , Baker Sherry M. , Brunmark Anders , Ling Ping , Darrow Andrew L. , Santulli Rosemary J. , Brass Lawrence F. , Andrade–Gordon Patricia
Published: April 2011

PAR-2 is a second member of a novel family of G-protein-coupled receptors characterized by a proteolytic cleavage of the amino terminus, thus exposing a tethered peptide ligand that autoactivates the receptor. The physiological and/or pathological role(s) of PAR-2 are still unknown. This study provides tissue-specific cellular localization of PAR-2 in normal human tissues by immunohistochemical techniques. A polyclonal antibody, PAR-2C, was raised against a peptide corresponding to the amino terminal sequence SLIGKVDGTSHVTGKGV of human PAR-2. Significant PAR-2 immunoreactivity was detected in smooth muscle of vascular and nonvascular origin and stromal cells from a variety of tissues. PAR-2 was also present in endothelial and epithelial cells independent of tissue type. Strong immunolabeling was observed throughout the gastrointestinal tract, indicating a possible function for PAR-2 in this system. In the CNS, PAR-2 was localized to many astrocytes and neurons, suggesting involvement of PAR-2 in neuronal function. A role for PAR-2 in the skin was further supported by its immunolocalization in the epidermis. PAR-2C antibody exemplifies an important tool to address the physiological role(s) of PAR-2.
The Use of Avidin as a Probe for the Distribution of Mitochondrial Carboxylases in Developing Chick Retina
Authors: Ruggiero Francis P. , Sheffield Joel B.
Published: April 2011

During development, the inner chick retina progresses from an aerobic to an anaerobic metabolic basis because of the lack of a vascular system. To investigate this process further, we have examined the expression and distribution of mitochondrial carboxylases. Because these enzymes use covalently bound biotin as a co-enzyme, we were able to develop a new detection protocol for mitochondria using avidin as a probe for the biotin. Chemiluminescent detection of bound avidin–peroxidase was used to examine a developmental series of extracts of retinas that had been separated by electrophoresis and blotted to nitrocellulose. Avidin–peroxidase, visualized with the sensitive peroxidase substrate True Blue, permitted detection in epoxy-embedded tissue sections. In the extracts, specific bands of approximate molecular weights 130 and 70 kD were found, corresponding to biotinylated subunits of several mitochondrial carboxylases. During development, the intensity of the bands decreases, although at different rates. In tissue sections, 8-day embryonic retinas display reaction product throughout the tissue, with higher local concentrations in the vitread and sclerad regions. During further development, the reaction product becomes segregated into bands at the borders of the plexiform layers. As the photoreceptors mature, stain becomes concentrated in the developing ellipsoids and the sclerad ends of Müller cells.
Expression and Distribution of Heme Oxygenase-2 mRNA and Protein in Rat Kidney
Authors: Hu Ying , Ma Ning , Yang Miao , Semba Reiji
Published: April 2011

Recent studies suggest that carbon monoxide (CO), which is formed by the enzyme heme oxygenase (HO) during the conversion of heme to biliverdin, shares some of the chemical and biological properties of nitric oxide (NO) and may play roles similar to those of NO. Heme oxygenase activity in the kidney has been reported for many years, and there are some reports on the expression of mRNA for two HO isozymes (HO-1 and HO-2) and cellular localization of HO-1 protein. However, cellular localization of HO-2 protein in the kidney under normal conditions has not been reported. In the present study we examined the expression and distribution of HO-2 mRNA and HO-2 protein in rat kidney using RNA protection assay and light and electron immunocytochemistry. RNA protection assay confirmed constitutive expression of HO-2 transcript in rat kidney. HO-2 immunoreactivity was selectively found in epithelial cells of the thick ascending limb and distal convoluted tubule, connecting tubule cells, and principal cells of the collecting duct. These results suggest that HO-2 is synthesized in the kidney and that HO-2 in the epithelial cells of renal tubules may serve as a source for CO generation under normal conditions.
Evaluation of the Sensitivity of the Terminal Deoxynucleotidyl Transferase–Immunogold Technique on Balbani Ring Genes
Authors: Thiry Marc , Daneholt Bertil
Published: April 2011

Recently, we developed the terminal deoxynucleotidyl transferase (TdT)–immunogold technique for in situ detection of DNA molecules. In this study the potential value and the limitations of the method were evaluated using the giant polytene chromosomes from Chironomus tentans salivary glands. Emphasis was put on the Balbiani rings (BRs), specialized chromosomal sites with exceptionally intense synthesis of large mRNA molecules. Immunolabeling was recorded not only over the bands and interbands of the polytene chromosomes but also over the BR structures. In the BRs, gold particles were present over segments of active transcription units, each with a central chromatin axis and a number of growing RNP products attached to the axis. One third of the transversely sectioned transcription units showed labeling in the central parts, i.e., where the unfolded chromatin axis is located, whereas the growing RNP fibers remained unlabeled. The absence of labeling of the RNP fibers is not likely to be due to lack of accessibility, because anti-RNA antibodies readily decorated the RNP fibers. The nuclear sap and cytoplasm displayed no significant label. These results clearly indicate that the TdT–immunogold technique is specific for DNA and detects not only DNA in compacted chromatin but also fully extended DNA. Its ability to efficiently label a single DNA molecule demonstrates the method's very high sensitivity.
Distribution of Dystroglycan in Normal Adult Mouse Tissues
Authors: Durbeej Madeleine , Henry Michael D. , Ferletta Maria , Campbell Kevin P. , Ekblom Peter
Published: April 2011

Dystroglycan is a cell surface protein which, in muscle, links the extracellular matrix protein laminin-2 to the intracellular cytoskeleton. Dystroglycan also binds laminin-1 and the binding occurs via the E3 fragment of laminin-1. Recently, it was found that dystroglycan is expressed in developing epithelial cells of the kidney. Moreover, antibodies against dystroglycan can perturb epithelial development in kidney organ culture. Therefore, dystroglycan may be an important receptor for cell–matrix interactions in non-muscle tissues. However, information about the tissue distribution of dystroglycan is limited, especially in adult tissues. Here we show that dystroglycan is present in epithelial cells in several non-muscle organs of adult mice. Dystroglycan is enriched towards the basal side of the epithelial cells that are in close contact with basement membranes. We suggest that dystroglycan is involved in linking basement membranes to epithelial and muscle cells. Dystroglycan may be important for the maintenance of tissue integrity.
1,25-Dihydroxy Vitamin D3 and Tri-iodothyronine Stimulate the Expression of a Protein Immunologically Related to Osteocalcin
Authors: Luegmayr Eva , Varga Franz , Glantschnig Helmut , Fratzl–Zelman Nadja , Rumpler Monika , Ellinger Adolf , Klaushofer Klaus
Published: April 2011

Osteocalcin (OC), a bone-specific protein, is a marker of late osteoblastic differentiation. Its expression is influenced by various growth factors and hormones. We investigated the effect of 1,25-dihydroxy vitamin D3 (D3) and tri-iodothyronine (T3) on OC expression in osteoblast-like MC3T3-E1 cells. A heterologous OC green fluorescence protein (GFP) fusion vector was established and expressed to study possible effects on protein transport. Immunostaining of endogenous OC revealed a significant increase in the percentage of positive cells after D3 and T3 treatment. This was consistent for MC3T3-E1 cells as well as nonosteogenic NIH-3T3 and mammary carcinoma cells, but not for neuroblastoma cells. The perinuclear immunostaining corresponded to the NBD C6 ceramide Golgi staining. Conversely, we found a strong induction of OC in MC3T3-E1 cells at the mRNA and protein levels only with T3 and not with D3. OC mRNA and protein expression was not detected in NIH fibroblasts. OC GFP transfection experiments indicate rapid transport and secretion of OC, because OC GFP was not found to be accumulated at intracellular compartments after hormone treatment. We conclude that the strong perinuclear immunostaining does not represent OC but a protein immunologically related to OC, as indicated by preabsorption experiments. The expression of this OC epitope-sharing protein is regulated by both D3 and T3 in the osteoblastic MC3T3-E1 and in nonosteogenic cells.
Cellular Localization of Calmodulin-dependent Protein Kinases I and II to A-cells and D-cells of the Endocrine Pancreas
Authors: Matovcik Lisa M. , Nairn Angus C. , Gorelick Fred S.
Published: April 2011

Ca2+/calmodulin-dependent protein kinases I and II, initially identified in brain on the basis of their ability to phosphorylate synapsin I, have been implicated in the regulation of Ca2+-dependent synaptic neurosecretion. Specific recombinant and synthetic peptide antibodies were used to examine the distribution of CaM kinases I and II in the rat pancreas and other tissues. The CaM kinase I antibodies detected a doublet of cytosolic proteins of ∼38 and ∼42 kD by immunoblot. CaM kinase I was observed in glucagon-containing A-cells at the periphery of the islet of Langerhans but had little or no overlap with pancreatic polypeptide or somatostatin cells. In contrast, CaM kinase II was localized to somatostatin-containing D-cells. CaM kinase I co-localized with glucagon secretory granules. CaM kinase II was not associated with the somatostatin granule but rather was enriched in areas of the cells that contained relatively little somatostatin. Because glucagon secretion is Ca2+-dependent, it is attractive to speculate that CaM kinase I may play a regulatory role in glucagon secretion. Glucagon and somatostatin cells both utilize intracellular Ca2+ for signaling. Therefore, specific CaM kinases may act as effectors of Ca2+ in these different cell types.
Vascular Permeability Factor/Vascular Endothelial Growth Factor and Vascular Stroma Formation in Neoplasia: Insights from In Situ Hybridization Studies
Authors: Brown Lawrence F. , Guidi Anthony J. , Tognazzi Kathi , Dvorak Harold F.
Published: April 2011

The formation of vascular stroma plays an important role in the pathophysiology of malignancy. We describe the use of in situ hybridization in our laboratory as a tool to study the role of vascular permeability factor/vascular endothelial growth factor in the angiogenesis associated with malignancy.
Putative Preneoplastic Changes Identified by Enzyme Histochemical and Immunohistochemical Techniques
Authors: Pretlow Theresa P. , Pretlow Thomas G.
Published: April 2011

Microscopic evaluation of whole-mount colons stained with methylene blue and/or hexosaminidase has identified putative preneoplastic lesions in the colons of rodents treated with carcinogen and in the grossly normal colons of humans. Enzyme histochemistry with glycol methacrylate sections has permitted the identification of putative premalignant lesions in rodent livers, human and rodent colons, and human prostates. Immunohistochemistry with paraffin-embedded tissues has been used to identify and characterize putative premalignant lesions in human colons and prostates.
Insights About Brain Tumors Gained Through Immunohistochemistry and in Situ Hybridization of Nuclear and Phenotypic Markers
Authors: McKeever Paul E.
Published: April 2011

Immunohistochemistry (IHC) has provided major insights about the classification of brain tumors by identifying cellular markers of phenotype and about tumor growth potential with nuclear markers of proliferation. In situ hybridization (ISH) research shows promise for diagnostic applications in tumor classification. The avidin-biotin conjugate IHC procedure is highlighted for diagnostic use on routinely processed clinical specimens. The immunophenotypes of brain tumors are tabulated in reference to their common IHC markers. Tumors that have been correctly classified by their IHC phenotypes include the giant-cell glioblastoma, primary brain lymphoma, and central neurocytoma. Phenotypes that may be more definitively detected by ISH, such as pituitary hormone, immunoglobulin light chain, and collagen messages are described. IHC of nuclear proliferation markers correlates with grade of malignancy, predicts tumor growth potential, and is prognostic for patient survival. The incorporation of bromodeoxyuridine, the expression of proliferating cell nuclear antigen, and the expression of Ki-67 antigen detected by MIB-1 antibody are compared in regard to their cell cycle activity and labeling index determinations. Fluorescence in situ hybridization (FISH) of brain tumor interphase nuclei and chromosomes is described. Abnormal FISH signals of specific chromosomes are associated with different types of brain tumors, with different grades of malignancy, and with mesenchymal drift of glioma cells in culture.
Preferential Activation of the Epidermal Growth Factor Receptor in Human Colon Carcinoma Liver Metastases in Nude Mice
Authors: Parker Craig , Roseman Barry J. , Bucana Corazon D. , Tsan Rachel , Radinsky Robert
Published: April 2011

Increased epidermal growth factor receptor (EGF-R) gene expression and functional protein levels correlate with the metastatic potential of human colon carcinoma (HCC) cells in nude mice. The purpose of this study was to determine whether the production of liver metastases by HCC cells depends on the EGF-R activation status and whether different organ microenvironments influence this activation. Using two independent monoclonal antibodies specific for the activated (i.e., tyrosine-phosphorylated) EGF-R, increased immunoreactivity was observed in HCC cells growing as metastatic lesions in the livers of athymic nude mice. Staining was observed throughout these lesions, both peripherally and centrally. In contrast, little or no immunoreactivity for activated EGF-R was observed in primary tumors growing orthotopically in the cecum or ectopically in the subcutis of nude mice. Immunohistochemistry for total EGF-R levels (irrelevant of activation status) demonstrated similar levels of immunoreactivity in HCC tumors growing in the cecum, subcutis, or liver of nude mice, indicating that total EGF-R levels are not altered after growth in these different microenvironments. Controls included immunohistochemistry for total and activated EGF-R levels in HCC cells growing in vitro under serum-free or EGF-stimulated conditions and A431-epidermoid carcinoma growing in nude mice. Western blot analyses confirmed the specificity of the antibodies for the activated EGF-R. These results suggest that the production of liver metastasis by HCC cells depends in part on the response of tumor cells to organ-derived growth factors and hence the activation of specific cell surface tyrosine kinase receptors.
Endogenous IL-2 in Cancer Cells: A Marker of Cellular Proliferation
Authors: Reichert Torsten E. , Watkins Simon , Stanson Joanna , Johnson Jonas T. , Whiteside Theresa L.
Published: April 2011

We have previously demonstrated that interleukin-2 (IL-2) receptors, IL-2 protein, and mRNA for IL-2 are present in human carcinomas in vitro and in vivo. Carcinoma cells synchronized in the G2/M-phase of the cell cycle express significantly more intracytoplasmic IL-2 as well as IL-2R-β and -γ than tumor cells in the G0/G1-phase. Here we evaluated immunohistologically the cell cycle-dependent distribution of the proliferation-associated Ki-67 antigen and expression of the cytokine IL-2 in four different carcinoma cell lines. In addition, 34 tissue samples from patients with squamous cell carcinomas of the head and neck were simultaneously analyzed for Ki-67 and IL-2 expression and the data were correlated to the histological grade of the tumors. All tumor cell lines were shown to express IL-2 in the Golgi complex. The strongest IL-2 expression was seen in tumor cells undergoing mitosis, identified by double staining with the antibody to Ki-67. In the tumor tissue, the highest level of co-expression of IL-2 and Ki-67 was observed in poorly differentiated carcinomas, with a labeling index (LI) of 67.2% for IL-2 and 68.8% for Ki-67. Well-differentiated carcinomas showed a significantly lower expression of both proteins (LI 35.0% for IL-2 and 26.5% for Ki-67). The correlation between the labeling indices was statistically significant ( r = 0.747; p<0.001). These results demonstrate that IL-2 expression in human carcinoma tissues is strongly associated with cell proliferation and significantly correlates with the histological tumor grade.
Lectin Intravital Perfusion Studies in Tumor-bearing Mice: Micrometer-resolution, Wide-area Mapping of Microvascular Labeling, Distinguishing Efficiently and Inefficiently Perfused Microregions in the Tumor
Authors: Debbage Paul L. , Griebel Jurgen , Ried Monika , Gneiting Thomas , DeVries Alexander , Hutzler Peter
Published: April 2011

Intravital lectin perfusion was combined with computer-guided scanning digital microscopy to map the perfused elements of the vasculature in tumor-bearing mice. High-precision composite images (spatial precision 1.3 μm and optical resolution 1.5 μm) were generated to permit exact positioning, reconstruction, analysis, and mapping of entire tumor cross-sections (c. 1 cm in diameter). Collation of these mosaics with nuclear magnetic resonance maps in the same tumor plane identified sites of rapid contrast medium uptake as tumor blood vessels. Digitized imaging after intravital double labeling allowed polychromatic visualization of two different types of mismatched staining. First, simultaneous application of two lectins, each bearing a different fluorochrome, revealed organ-specific differential processing in the microvascular wall. Second, sequential application of two boluses of one lectin, bearing different fluorochromes successively, distinguished between double-labeled microvessels, representing efficiently perfused vascular segments, and single-labeled microvessels, with inefficient or intermittent perfusion. Intravital lectin perfusion images of blood vessels in the vital functional state thus highlighted biologically significant differences in vessel function and served as high-resolution adjuncts to MR imaging.
ADAM-10 Message Is Expressed in Human Articular Cartilage
Authors: Chubinskaya Susan , Cs-Szabo Gabriella , Kuettner Klaus E.
Published: April 2011

The depletion of the pericellular and territorial matrices in articular cartilage is considered to be one of the earliest events in pathobiology of osteoarthritis (OA). A newly discovered family of proteins with a disintegrin-like and metalloproteinase-like domain (ADAM) may be involved in matrix degradation as well as in cell-cell and cell-matrix interactions. The purpose of this study was to determine by in situ hybridization whether human articular chondrocytes from newborn, normal adult, and OA cartilages express messenger RNA for ADAM-10, one of the members of this family, and by semiquantitative RTPCR to compare the levels of this expression. The results confirmed the expression of ADAM-10 by human articular chondrocytes and revealed the highest levels of expression in the continuously remodeling cartilage of newborns and the most fibrillated areas of OA cartilage, especially the regions of cell clusters. Importantly, ADAM-10 mRNA expression was evident in tissues with the greatest loss of Safranin O staining from the territorial and interterritorial matrix of the chondrocytes. Messenger RNA was upregulated in OA tissue compared to the age-matched normal cartilage, as detected by RT-PCR. Upregulated levels of ADAM-10 mRNA expression appear to be related to the degree of cartilage damage and/ or degradation, which suggests a potential role for at least one member of this new family in the cartilage matrix destruction accompanying OA.
Placental Lactogen-I (PL-I) Target Tissues Identified with an Alkaline Phosphatase-PL-I Fusion Protein
Authors: Müller Heiner , Dai Guoli , Soares Michael J.
Published: April 2011

The rat placenta expresses a family of genes related to prolactin (PRL). Target tissues and physiological roles for many members of the PRL family have yet to be determined. In this investigation we evaluated the use of an alkaline phosphatase (AP) tag for monitoring the behavior of a prototypical member of the PRL family, placental lactogen-I (PL-I). A probe was generated consisting of a fusion protein of human placental AP and rat PL-I (AP-PL-I). The AP-PL-I construct was stably expressed in 293 human fetal kidney cells, as was the unmodified AP vector that served as a control. AP activity was monitored with a colorimetric assay in conditioned medium from transfected cells. Immunoreactivity and PRL-like biological activities of the AP-PL-I fusion protein were demonstrated by immunoblotting and the Nb2 lymphoma cell proliferation assay, respectively. AP-PL-I specifically bound to tissue sections known to express the PRL receptor, including the ovary, liver, and choroid plexus. Binding of AP-PL-I to tissues was specific and could be competed with ovine PRL. The results indicate that AP is an effective tag for monitoring the behavior of PL-I and suggest that this labeling system may also be useful for monitoring the actions of other members of the PRL family.
Differential Localization of Cysteine Protease Inhibitors and a Target Cysteine Protease, Cathepsin B, by Immuno-Confocal Microscopy
Authors: Calkins Cathárine C. , Sameni Mansoureh , Koblinski Jennifer , Sloane Bonnie F. , Moin Kamiar
Published: April 2011

The cystatin superfamily of cysteine protease inhibitors and target cysteine proteases such as cathepsin B have been implicated in malignant progression. The respective cellular/extracellular localization of cystatins and cysteine proteases in tumors may be critical in regulating activity of the enzymes. Confocal microscopy has enabled us to demonstrate the differential localization of cystatins and cathepsin B in an embryonic liver cell line and an invasive hepatoma cell line. In both, stefins A and B were distributed diffusely throughout the cytoplasm, whereas cystatin C was distributed in juxtanuclear vesicles. Stefin A and cystatin C, but not stefin B, were present on the cell surface. Cystatin C was found on the top surfaces of both cell lines, whereas stefin A was found only on the top surface of the embryonic liver cells. Cathepsin B staining was concentrated in perinuclear vesicles in the embryonic liver cells. In the hepatoma cells, staining for cathepsin B was also present in vesicles adjacent to the cell membrane and on localized regions of the bottom surface. Such a disparate distribution of cathepsin B and its endogenous inhibitors may facilitate proteolysis by the hepatoma cells and thereby contribute to their invasive phenotype.
An In Situ Hybridization Histochemistry Technique Allowing Simultaneous Visualization by the Use of Confocal Microscopy of Three Cellular mRNA Species in Individual Neurons
Authors: Grino Michel , Zamora Alfredo J.
Published: April 2011

We present a specific and sensitive method for simultaneous detection of three mRNA species in individual neurons. The method relies on the use of riboprobes labeled with [35S]-UTP, digoxigenin-UTP, or biotin-UTP. The nonradioactive probes were sequentially revealed by incubation with anti-digoxigenin immunoglobulins or streptavidin conjugated to peroxidase, followed by the use of fluorochrome-labeled tyramides as peroxidase substrates. The radioactive probe was revealed by conventional autoradiography. There was no interaction among the different probes or the various detection systems. We demonstrate the use of this method by illustrating on laser scanning confocal microscopy the co-localization of the mRNAs coding for corticotropin-releasing factor (CRF), arginine vasopressin (AVP), or peptidylglycine α-amidating monooxygenase (PAM) in rat hypothalamic paraventricular nucleus (PVN) and its modulation by endogenous glucocorticoids. Our results suggest that this method could be used not only to study the regulation of the hypothalamo-pituitary-adrenal axis but also in various models in which mRNAs are present at low concentrations.
Flow Cytometric Assay of Cytochemically Demonstrated NAD(P)H Oxidoreductase (Diaphorase) Activities
Authors: Severin Erhard , Seidler Eberhard
Published: April 2011

The tetrazolium salt 5-cyano-2,3-di- p-toluyl-tetrazolium chloride (CTC), yielding a fluorescent formazan on reduction, was used to measure NAD(P)H oxidoreductase activity. In this study, optimal conditions for the flow cytometric technique were determined empirically with tissue culture cell lines and mouse Ehrlich ascites cells. Applying a coupled reaction procedure, NADH and NADPH as substrates of the oxidoreductases to be measured are generated endogenously by lactate or glucose-6-phosphate dehydrogenase, respectively. The results were evaluated by combining spectrophotometry and flow cytometry. We obtained integral activities for each group of NADH and NADPH oxidoreductases. Furthermore, by counterstaining the DNA with DAPI, followed by bivariate analysis of flow cytometric data, our assay gives a detailed distribution of enzyme activities of all cells, even in subgroups present in heterogeneous cell populations. Therefore, this protocol permits the study of NAD(P)H oxidoreductase activities in ex vivo tumor samples in which mixed cellular populations may be present.
Rapid Synthesis of Biotin-, Digoxigenin-, Trinitrophenyl-, and Fluorochrome-labeled Tyramides and Their Application for In Situ Hybridization Using CARD Amplification
Authors: Hopman Anton H.N. , Ramaekers Frans C.S. , Speel Ernst J.M.
Published: April 2011

A one-step procedure for the synthesis of different tyramide conjugates, which can be utilized in the catalyzed reporter deposition (CARD) amplification system, is described. Succinimidyl esters of biotin, digoxigenin, and of the fluorochromes fluorescein, rhodamine, aminomethylcoumarine acetic acid, and Cy3 were coupled to tyramine in dimethylformamide (DMF) adjusted to a pH of 7.0–8.0 with triethylamine (TEA). The coupling reaction can be performed within 2 hr and the reaction mixture can be applied without further purification steps. Furthermore, trinitrophenyl (TNP)-tyramide was prepared by adding 2,4,6,-trinitrobenzenesulfonic acid to tyramine dissolved in either MilliQ/DMF basified with TEA or in an NaHCO3 (pH 9.5) buffer. A subsequent precipitation of the TNP-tyramide resulted in a high-yield isolation of this conjugate. The synthesized tyramide conjugates were applied successfully in single- and multiple-target in situ hybridization (ISH) procedures to detect both repetitive and single-copy DNA target sequences in cell preparations with high efficiency. The described approach provides an easy and fast method to prepare a variety of tyramide conjugates in bulk amounts at relatively low cost.
NADPH Diaphorase Histochemistry Detects Inducible Nitric Oxide Synthetase Activity in the Thymus of Naive and Staphylococcal Enterotoxin B-stimulated Mice
Authors: Virág László , Haskó György , Salzman Andrew L. , Szabó Csaba
Published: April 2011

Here we examined the changes in NADPH diaphorase (NADPHd) and inducible nitric oxide synthetase (iNOS) positivity in the medulla of the mouse thymus in response to treatment with the superantigen, Staphylococcal enterotoxin B (SEB). A few NADPHd+ and iNOS+ cells scattered in the medulla were detected in the thymi of naive mice. SEB induced the appearance of a large number of NADPHd+- and iNOS-immunoreactive cells in the thymic medulla. In the thymus of iNOS-deficient mice, a total absence of these NADPHd+ and iNOS+ medullary cells was found both under basal conditions and after SEB stimulation. With the NADPHd reaction, only endothelial staining was detected in the thymi of iNOS-deficient mice. Our data indicate that NADPHd+ cells in the thymic medulla express iNOS and that SEB induces iNOS expression in the mouse thymus.
Histochemical Reactivity of Normal, Metaplastic, and Neoplastic Tissues to α-Linked N-Acetylglucosamine Residue-specific Monoclonal Antibody HIK1083
Authors: Nakamura Naoshi , Ota Hiroyoshi , Katsuyama Tsutomu , Akamatsu Taiji , Ishihara Kazuhiko , Kurihara Makoto , Hotta Kyoko
Published: April 2011

Monoclonal antibody (MAb) HIK1083, which is obtained by immunizing mice with a preparation of rat gastric mucins, has been shown to bind specifically to α-linked N-acetylglucosamine (α-GlcNAc). We investigated the specificity of MAb HIK1083 by immunostaining normal human organs, mucinous metaplasia of human pancreas, adenocarcinomas of human stomach, pancreas, and colon, and normal rat organs. The specificity was investigated by making comparisons with (a) a stain that labels Class III concanavalin A (ConA)-reactive mucin (Class III mucin), i.e., paradoxical ConA (PCS), and (b) staining with horseradish peroxidase (HRP)-conjugated Griffonia simplicifolia agglutinin II (GSA-II). In normal human and rat organs and in mucinous metaplasia of human pancreas, immunostaining with MAb HIK1083 and PCS showed similar specificities for mucins in glandular mucous cells. In adenocarcinoma of stomach and pancreas, GSA-II showed the most widespread positivity, PCS showed the least, and MAb HIK1083 showed a reactivity between those two extremes. Colon adenocarcinomas were labeled only with GSA-II. These results demonstrate that MAb HIK1083 could be a useful screening tool for Class III mucin in normal, metaplastic, and carcinoma tissues, and that the α-GlcNAc residue is one of the specific sugar residues found in Class III mucin.
Detection of Deoxyribonuclease I Along the Secretory Pathway in Paneth Cells of Human Small Intestine
Authors: Shimada Osamu , Ishikawa Harunori , Tosaka-Shimada Hisami , Yasuda Toshihiro , Kishi Koichiro , Suzuki Shosuke
Published: April 2011

The expression and distribution of deoxyribonuclease I (DNase I) in human duodenum, jejunum and ileum were examined by DNase I activity assay and the reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, in situ hybridization, and immunocytochemical ultrastructural analyses. High levels of DNase I were detected in the cytoplasm of Paneth cells in human small intestine. A tissue homogenate fraction rich in Paneth cells showed strong DNase I-specific enzymatic activity. Immunofluorescence analysis using several specific anti-human DNase I antibodies showed very strong immunoreactivity in the cytoplasm of every Paneth cell. In situ hybridization demonstrated high levels of DNase I mRNA in Paneth cells. Immunogold electron microscopy revealed gold particles localized along the secretory pathway, with the exocrine secretory granules mostly labeled. Our findings strongly suggest that Paneth cells synthesize and secrete DNase I into the intestinal lumen.
Optimized Fixation and Storage Conditions for FISH Analysis of Single-cell Suspensions
Authors: Murrell-Bussell Sarah , Nguyen Dianne , Schober Wendy D. , Scott Jeffrey , Simpson Joe Leigh , Elias Sherman , Bischoff farideh Z. , Lewis Dorothy E.
Published: April 2011

SUMMARY In our protocol to isolate and identify fetal cells in maternal peripheral blood, antibody (Ab)-stained cells are preserved with paraformaldehyde (PF) before batch flow cytometric sorting. However, PF fixation compromises the quality of subsequent interphase fluorescence in situ hybridization (FISH). We therefore examined the effect of PF concentrations and storage time in phosphate-buffered saline (PBS) on the quality of FISH signals. Cells were analyzed for changes in light scatter, morphology, and accessibility of target cell DNA. Fixation in 3% PF for 1 hr was ideal for both flow cytometry and subsequent FISH detection. However, beyond 10 days of storage, FISH quality deteriorated.