scholarly journals Double labeling of SIgG+ cells harboring a lymphotropic herpesvirus by avidin-biotin complex immunoperoxidase and immunogold-silver staining techniques.

1988 ◽  
Vol 36 (9) ◽  
pp. 1187-1189 ◽  
Author(s):  
Z Nagy-Oltvai ◽  
T Brady ◽  
G D Hsiung
Keyword(s):  
Cell Surface ◽  
Silver Staining ◽  
Simultaneous Detection ◽  
Double Labeling ◽  
Tissue Sections ◽  
Surface Immunoglobulin ◽  
Staining Techniques ◽  
Double Immunolabeling ◽  
Usual Order ◽  
Avidin Biotin Complex

By reversing the usual order of double-staining procedures, we obtained optimal conditions for simultaneous detection of guinea pig lymphotropic herpesvirus (GPHLV) and surface immunoglobulin G-positive (SIgG+) cells in paraffin-embedded tissue sections. Of the different fixatives tested, Bouin's solution gave the best preservation of morphology and cell surface IgG. Double immunolabeling was best achieved when avidin-biotin complex immunoperoxidase (ABC-IP) staining was performed first for detection of intracellular viral antigen, followed by immunogold-silver staining (IGSS) for localization of cell surface IgG.

scholarly journals Double-immunolabeling systems for phenotyping of immune cells harboring bovine viral diarrhea virus.

1987 ◽  
Vol 35 (6) ◽  
pp. 627-633 ◽  
Author(s):  
H Bielefeldt Ohmann
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Simultaneous Detection ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Tissue Sections ◽  
Specificity And Sensitivity ◽  
Infected Cells ◽  
Viral Diarrhea Virus ◽  
Double Immunolabeling

Optimal staining conditions were defined for simultaneous detection of bovine viral diarrhea virus (BVDV) and mononuclear leukocyte surface antigens in tissue sections and cytospins. Because of the extreme lability of the virus antigens and the variable stability of the epitopes on the cell differentiation antigens, cryopreservation had to be used. This method gives slightly sub-optimal preservation of morphology. However, the specificity and sensitivity of the immunolabeling ensured reliable identification of the double-labeled cells, i.e., the phenotypic identification of virus-infected cells within the immune system.

scholarly journals An immunoenzyme triple-staining method using both polyclonal and monoclonal antibodies from the same species. Application of combined direct, indirect, and avidin-biotin complex (ABC) technique.

1987 ◽  
Vol 35 (11) ◽  
pp. 1199-1204 ◽  
Author(s):  
C M van der Loos ◽  
P K Das ◽  
H J Houthoff
Keyword(s):  
Monoclonal Antibodies ◽  
Staining Method ◽  
Polyclonal Antibodies ◽  
Simultaneous Detection ◽  
Reaction Products ◽  
Tissue Sections ◽  
Beta Galactosidase ◽  
Immunohistological Analysis ◽  
Triple Staining ◽  
Avidin Biotin Complex

For immunohistological analysis, simultaneous detection of multiple cellular epitopes, as compared to single staining of serial sections, is sometimes needed. Therefore, immunoenzyme triple-staining protocols were tested with polyclonal and monoclonal antibodies on tissue sections and cytospin preparations. Various immunoconjugates were used in different combinations of methods, of which not all proved to be suitable. Of the tested protocols, one yielded superior results for both monoclonal and polyclonal antibodies, with optimal preservation of their original avidity. The method consists of a combination of indirect, direct, and avidin-biotin complex technique. The three antigens can be distinguished clearly and selectively by the reaction products of the enzyme activities of beta-galactosidase (green), alkaline phosphatase (blue), and horseradish peroxidase (red).

scholarly journals CELL SURFACE IMMUNOGLOBULIN

1972 ◽  
Vol 135 (6) ◽  
pp. 1392-1405 ◽  
Author(s):  
Charles J. Sherr ◽  
Sonia Baur ◽  
Inge Grundke ◽  
Joseph Zeligs ◽  
Barbara Zeligs ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Noncovalent Interactions ◽  
Subcellular Fractionation ◽  
Splenic Lymphocytes ◽  
Surface Immunoglobulin ◽  
Established Line ◽  
Intracellular Proteins ◽  
Daudi Cells

Cells from an established line of Burkitt lymphoma (Daudi) were enzymatically radioiodinated, and labeled Ig from the cell surface was isolated and studied. Subcellular fractionation of labeled cells confirmed that intracellular proteins from the cytoplasm are not iodinated by this method. Radioactive Ig was identified as monomeric (8S) IgM, and an average of 105 Ig molecules was found per cell. Ig molecules could be released from the plasma membrane by detergent lysis under nonreducing conditions indicating that attachment of Ig to the plasma membrane occurs via noncovalent interactions. The ratio of µ/L radioactivity in surface Ig was the same as that of total cellular Ig radioiodinated in solution suggesting that a large portion of the Fc fragment is not buried within the membrane. In contrast to the results obtained with cell surface Ig, most intracellular Ig was found as "free" µ- and L chains regardless of whether lysates were labeled with 125I or cells were labeled with leucine-3H. The results indicate that only a small percentage of the total Ig of Daudi cells is associated with the cell surface and suggest that covalent assembly of Ig occurs at or near the time that the molecule becomes part of the plasma membrane. Similarities between cell surface Ig on normal splenic lymphocytes and Daudi cells suggest that the latter is a neoplasm of bone marrow-derived lymphocytes.

Surface immunoglobulin restriction in B-non Hodgkin's lymphomas in cell suspension and on frozen tissue sections

2009 ◽  
Vol 35 (4) ◽  
pp. 399-407 ◽  
Author(s):  
Philip M. Kluin ◽  
Roel A. Weger ◽  
Henk-Jan Schuurman ◽  
Peter P. J. Peters ◽  
Philomé I. Spies ◽  
...  
Keyword(s):  
Cell Suspension ◽  
Tissue Sections ◽  
Surface Immunoglobulin ◽  
Frozen Tissue ◽  
Hodgkin's Lymphomas

Functional partitioning of beta1 integrins revealed by activating and inhibitory mAbs

1997 ◽  
Vol 110 (21) ◽  
pp. 2647-2659 ◽  
Author(s):  
M.T. Cruz ◽  
C.L. Dalgard ◽  
M.J. Ignatius
Keyword(s):  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Dermal Fibroblasts ◽  
The Other ◽  
Embryonic Chick ◽  
Cell Surfaces ◽  
Double Labeling ◽  
Focal Contacts ◽  
Functional Partitioning ◽  
Discrete Populations

Integrins exist in different activation states on the surfaces of cells. Addition of the proper signal, ligand, or antibody can alter the activation state of these molecules. We report here the identification of two immunocytochemically distinct populations of beta1 integrins on fixed embryonic chick dermal fibroblasts. One population, recognized by the integrin activating mAb TASC, localizes to discrete regions of the cell, most likely focal contacts. These integrins co-localize with other proteins, such as vinculin and F-actin, and their retention at these sites is dependent on the actin cytoskeleton. The other population, identified with the inhibitory mAb W1B10, is more evenly distributed throughout the cell surface, and its pattern remains unchanged after disruption of the actin cytoskeleton. Double labeling experiments using Fab fragments of TASC alongside whole W1B10 IgG revealed non-overlapping staining patterns. These results show that it is possible to visualize and study discrete populations of integrins on cell surfaces using two different antibodies. We hypothesize that these antibodies report differences in the distribution of receptors in two different states. A model is proposed describing the ligand independent recruitment of integrins based on these findings and results from other labs.

scholarly journals The Application of Immunogold Silver Staining (IGSS) for the Detection of Transmissible Gastroenteritis Virus in Fixed Tissues

1993 ◽  
Vol 5 (1) ◽  
pp. 16-20 ◽  
Author(s):  
Renke Larochelle ◽  
Ronald Magar
Keyword(s):  
Silver Staining ◽  
Protein A ◽  
Immunogold Electron Microscopy ◽  
Transmissible Gastroenteritis Virus ◽  
Tissue Sections ◽  
Gastroenteritis Virus ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Transmissible Gastroenteritis ◽  
Immune Aggregates

Protein A-gold (PAG) and a primary porcine antiserum were used in immunogold silver staining (IGSS) for the detection of transmissible gastroenteritis virus (TGEV) in formalin-fixed paraffin-embedded tissue sections of small intestine originating from infected pigs. Immunogold electron microscopy was used to evaluate the reactivity of the prepared PAG marker with the specific porcine TGEV antiserum. Gold particles were closely associated with single virions and immune aggregates of TGEV. When IGSS, using PAG as the marker, was applied to tissue sections, dark staining of TGEV-infected villous enterocytes was observed. Background was low, allowing good visualization by light microscopy of the distribution of viral antigen. Two other gold conjugates, protein A/G-gold (PA/GG) and protein G-gold (PGG), were tested in IGSS. The labeling with PA/GG was comparable to that obtained with PAG. However, no staining was observed when PGG was used. The use of IGSS and PAG offers advantages and may represent a useful technique for the detection of other viral pathogens.

Intracellular relationship between actin and alpha-actinin in a whole corneal epithelial tissue

1993 ◽  
Vol 106 (3) ◽  
pp. 703-717
Author(s):  
W. Khoory ◽  
E. Wu ◽  
K.K. Svoboda
Keyword(s):  
Cell Surface ◽  
Basal Cell ◽  
Laser Scanning ◽  
Cytochalasin D ◽  
Confocal Laser ◽  
Epithelial Tissue ◽  
Control Medium ◽  
Cell Nuclei ◽  
Double Labeling ◽  
Corneal Epithelial

Alpha-actinin is an actin crosslinking protein that may be one of the proteins involved in the attachment of the actin cytoskeletal framework to the plasma membrane. We investigated the distribution of alpha-actinin in whole-mount embryonic chick corneal epithelia using confocal laser scanning analysis. The intracellular alpha-actinin distribution was compared with F-actin using phalloidin, or total actin using an anti-actin antibody. Corneal epithelial tissues were isolated with or without the basal lamina (+ or -BL), and fixed immediately. In addition, epithelia isolated -BL were cultured for 2 hours with either control medium, laminin-supplemented medium or laminin and cytochalasin D (CD)-containing medium. The single- and double-labeled epithelia showed that alpha-actinin delineated the cell borders and microvilli of the periderm cells in the most apical optical sections of control and laminin-treated epithelia. At the optical plane through the basal cell nuclei, the alpha-actinin was distributed diffusely throughout the cytoplasm, whereas the actin was sparse, only associated with the lateral cell membranes. Epithelia (-BL) cultured in control medium had cytoplasmic protrusions or blebs on the basal cell surface. The blebs contained both actin and alpha-actinin. In epithelial cultured with laminin, the basal cell surface was flat. The actin cortical mat became reorganized within two hours. Actin and alpha-actinin were colocalized in the re-formed basal cytoskeletal network. In cells cultured with cytochalasin D (CD) and laminin the actin cortical mat was not reorganized. Actin networks from both cell layers were eliminated and replaced by aggregates scattered throughout the cytoplasm. The alpha-actinin remained diffusely distributed in the cytoplasm and failed to colocalize with the actin aggregates. The alpha-actinin appeared closer to the basal cell membrane than the actin in cross-sectional views of the tissue. Results from these double-labeling experiments confirmed the intimate association of alpha-actinin and actin in the laminin-stimulated actin cortical mat reorganization. This study is the first to demonstrate that CD-aggregated F-actin does not capture the alpha-actinin. The alpha-actinin appeared to remain diffuse throughout the cytoplasm and separate from F-actinin; however, there was some overlap with G-actin.

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