Haemolytic Efficiency of Rabbit IgG Anti-Forssman Antibody and its Augmentation by Anti-Rabbit IgG

Light microscopic immunohistochemistry based on the principle of capillary action staining is a widely used method to localize antigens. Capillary action immunostaining, however, has not been tested or applied to detect antigens at the ultrastructural level. The aim of this work was to establish a capillary action staining method for localization of intracellular antigens, using colloidal gold probes.Post-embedding capillary action immunocytochemistry was used to detect maternal IgG in the small intestine of newborn suckling piglets. Pieces of the jejunum of newborn piglets suckled for 12 h were fixed and embedded into LR White resin. Sections on nickel grids were secured on a capillary action glass slide (100 μm wide capillary gap, Bio-Tek Solutions, Santa Barbara CA, distributed by CMS, Houston, TX) by double sided adhesive tape. Immunolabeling was performed by applying reagents over the grids using capillary action and removing reagents by blotting on filter paper. Reagents for capillary action staining were from Biomeda (Foster City, CA). The following steps were performed: 1) wet the surface of the sections with automation buffer twice, 5 min each; 2) block non-specific binding sites with tissue conditioner, 10 min; 3) apply first antibody (affinity-purified rabbit anti-porcine IgG, Sigma Chem. Co., St. Louis, MO), diluted in probe diluent, 1 hour; 4) wash with automation buffer three times, 5 min each; 5) apply gold probe (goat anti-rabbit IgG conjugated to 10 nm colloidal gold, Zymed Laboratories, South San Francisco, CA) diluted in probe diluent, 30 min; 6) wash with automation buffer three times, 5 min each; 7) post-fix with 5% glutaraldehyde in PBS for 10 min; 8) wash with PBS twice, 5 min each; 9) contrast with 1% OSO4 in PBS for 15 min; 10) wash with PBS followed by distilled water for5 min each; 11) stain with 2% uranyl acetate for 10 min; 12) stain with lead citrate for 2 min; 13) wash with distilled water three times, 1 min each. The glass slides were separated, and the grids were air-dried, then removed from the adhesive tape. The following controls were used to ensure the specificity of labeling: i) omission of the first antibody; ii) normal rabbit IgG in lieu of first antibody; iii) rabbit anti-porcine IgG absorbed with porcine IgG.

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Immunocytochemical localization of desmin and vimentin in chick embryonic myocardial cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100159783 ◽

1990 ◽

Vol 48 (3) ◽

pp. 448-449

Author(s):

Yukiko Sugi

Keyword(s):

Sodium Methoxide ◽

Intermediate Filament Protein ◽

Chick Embryos ◽

Immunocytochemical Localization ◽

Myocardial Cells ◽

Immunofluorescence Study ◽

Immunofluorescent Localization ◽

Rabbit Igg ◽

Semithin Sections ◽

Filament Protein

In cultured skeletal muscle cells of chick, one intermediate filament protein, vimentin, is primarily formed and then synthesis of desmin follows. Coexistence of vimentin and desmin has been immunocytochemically confirmed in chick embryonic skeletal musclecells. Immunofluorescent localization of vimentin and desmin has been described in developing myocardial cells of hamster. However, initial localization of desmin and vimentin in early embryonic heart has not been reported in detail. By quick-freeze deep-etch method a loose network of intermediate filaments was revealed to exist surrounding myofibrils. In this report, immunocytochemical localization of desmin and vimentin is visualized in early stages of chick embryonic my ocardium.Chick embryos, Hamburger-Hamilton (H-H) stage 8 to hatch, and 1 day old postnatal chicks were used in this study. For immunofluorescence study, each embryo was fixed with 4% paraformaldehyde and embedded in Epon 812. De-epoxinized with sodium methoxide, semithin sections were stained with primary antibodies (rabbit anti-desmin antibody and anti-vimentin antibody)and secondary antibody (RITC conjugated goat-anti rabbit IgG).

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In vitro assembly of 2-D crystals from partially purified EA1 protein secreted by Bacillus anthracis strain Delta Sterne-1

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169110 ◽

1994 ◽

Vol 52 ◽

pp. 276-277

Author(s):

Mary Beth Downs ◽

Wilson Ribot ◽

Joseph W. Farchaus

Keyword(s):

Cell Wall ◽

Molecular Sieves ◽

Cell Envelope ◽

Single Species ◽

Supporting Cell ◽

Surface Layers ◽

Rabbit Igg ◽

In Vitro Assembly ◽

Covalently Linked

Many bacteria possess surface layers (S-layers) that consist of a two-dimensional protein lattice external to the cell envelope. These S-layer arrays are usually composed of a single species of protein or glycoprotein and are not covalently linked to the underlying cell wall. When removed from the cell, S-layer proteins often reassemble into a lattice identical to that found on the cell, even without supporting cell wall fragments. S-layers exist at the interface between the cell and its environment and probably serve as molecular sieves that exclude destructive macromolecules while allowing passage of small nutrients and secreted proteins. Some S-layers are refractory to ingestion by macrophages and, generally, bacteria are more virulent when S-layers are present.When grown in rich medium under aerobic conditions, B. anthracis strain Delta Sterne-1 secretes large amounts of a proteinaceous extractable antigen 1 (EA1) into the growth medium. Immunocytochemistry with rabbit polyclonal anti-EAl antibody made against the secreted protein and gold-conjugated goat anti-rabbit IgG showed that EAI was localized at the cell surface (fig 1), which suggests its role as an S-layer protein.

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An immunocytochemical study of maternal immunoglobulin localization in the suckling pig intestine

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010016217x ◽

1990 ◽

Vol 48 (3) ◽

pp. 922-923

Author(s):

László G. Kömüves ◽

Donna S. Turner ◽

Kathy S. McKee ◽

Buford L. Nichols ◽

Julian P. Heath

Keyword(s):

Sodium Ethoxide ◽

Intracellular Localization ◽

Protein A ◽

Tween 20 ◽

Intestinal Epithelial ◽

Immunocytochemical Study ◽

Fish Skin ◽

Newborn Piglets ◽

Rabbit Igg ◽

Fish Skin Gelatin

In this study we used colloidal gold probes to detect the intracellular localization of colostral immunoglobulins in intestinal epithelial cells of newborn piglets.Tissues were obtained from non-suckled newborn and suckled piglets aged between 1 hour to 1 month. Samples were fixed in 2.5 % glutaraldehyde, osmicated and embedded into Spurr’s resin. Thin (80 nm) sections were etched with 5% sodium ethoxide for 5 min, washed and treated with 4 % sodium-m-periodate in distilled water for 30 min. The sections were then first incubated with blocking buffer (2 % BSA, 0.25 % fish skin gelatin, 0.5 % Tween 20 in 10 mM Trizma buffer, pH=7.4 containing 500 mM NaCl) for 30 min followed by the immunoreagents diluted in the same buffer, 1 hr each. For the detection of pig immunoglobulins a rabbit anti-pig IgG antiserum was used followed by goat anti-rabbit IgG-Au10 or protein A-Au15 probes.

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Faculty Opinions recommendation of A toolbox of anti-mouse and anti-rabbit IgG secondary nanobodies.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732335575.793542390 ◽

2018 ◽

Author(s):

Laura Trinkle-Mulcahy

Keyword(s):

Rabbit Igg

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Interactions of C-reactive protein with the complement system. III. Complement-dependent passive hemolysis initiated by CRP.

Journal of Experimental Medicine ◽

10.1084/jem.142.5.1065 ◽

1975 ◽

Vol 142 (5) ◽

pp. 1065-1077 ◽

Cited By ~ 80

Author(s):

A.P. Osmand ◽

R.F. Mortensen ◽

Joan Siegel ◽

H. Gewurz

Keyword(s):

Guinea Pig ◽

Human Serum ◽

Complete System ◽

Gel Filtration ◽

C Reactive Protein ◽

Inflammatory Reactions ◽

Reactive Protein ◽

Rabbit Igg ◽

The Complement System ◽

Pig Serum

Interactions of CRP with various substrates in the presence of human serum have been shown to result in efficient activation of C components C1-C5. We now report the ability of CRP to initiate C-dependent hemolysis. For this purpose CRP was isolated by affinity chromatography using pneumococcal CPS and gel filtration; its purity was established by several criteria. Erythrocytes were coated with CPS (E-CPS) and passively sensitized with CRP. C-dependent lysis of these cells was observed upon the addition of suitably absorbed human serum, and the efficiency of hemolysis compared favorably with that initiated by rabbit IgG anti-CPS antibody. CRP also sensitized E-CPS for lysis by guinea pig C; partial lysis was seen when C4-deficient guinea pig serum was used, suggesting that CRP also shares with antibody the ability of CRP to fully activate the C system and provide further evidence for a role for CRP similar to that of antibody in the initiation and modulation of inflammatory reactions via the complete system.

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Electron Microscopic Immunogold Localization of Salivary Mucins MG1 and MG2 in Human Submandibular and Sublingual Glands

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540305100109 ◽

2003 ◽

Vol 51 (1) ◽

pp. 69-79 ◽

Cited By ~ 21

Author(s):

Marco Piludu ◽

Sean A. Rayment ◽

Bing Liu ◽

Gwynneth D. Offner ◽

Frank G. Oppenheim ◽

...

Keyword(s):

Salivary Glands ◽

Expression Patterns ◽

Cell Types ◽

Mucous Cells ◽

Electron Microscopic ◽

Thin Sections ◽

Dense Cores ◽

Duct Cells ◽

Rabbit Igg ◽

Lowicryl K4m

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.

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Circular dichroism studies on proteolytic cleavage fragments from rabbit IgG

Immunochemistry ◽

10.1016/0019-2791(77)90248-8 ◽

1977 ◽

Vol 14 (4) ◽

pp. 263-268 ◽

Cited By ~ 12

Author(s):

G.A. Stewart ◽

P.M. Johnson ◽

M.W. Barrett ◽

P.M. Scopes ◽

D.R. Stanworth

Keyword(s):

Circular Dichroism ◽

Proteolytic Cleavage ◽

Rabbit Igg ◽

Circular Dichroïsm

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SEM localization of cell-surface-associated fibronectin in the cranium of chick embryos utilizing immunolatex microspheres

Development ◽

10.1242/dev.80.1.175 ◽

1984 ◽

Vol 80 (1) ◽

pp. 175-195

Author(s):

Stephen Meier ◽

Christopher Drake

Keyword(s):

Basement Membranes ◽

Chick Embryos ◽

Preimmune Serum ◽

Fluorescent Staining ◽

Cell Surfaces ◽

Cell Soma ◽

Rabbit Igg ◽

Neural Ectoderm ◽

Cranial Neural Crest Cells ◽

Testicular Hyaluronidase

Fibronectin has been localized to basement membranes and cell surfaces with the light microscope by fluorescent staining of thick sections, and with the TEM by immunoperoxidase reaction. However, these methods are limited because it is difficult to appreciate the patterned distribution of fibronectin from sectioned material. We have developed a probe for fibronectin that facilitates its identification with the SEM. Our probe consists of two parts; the first component is a derivatized methacrylate microsphere 90 nm in diameter, linked to purified sheep anti-rabbit IgG. The second component is anti-fibronectin IgG raised in rabbits. Stage-3 to -12 chick embryos were fixed and the ectoderm covering the cranial mesoderm was removed. Embryos were treated with testicular hyaluronidase, exposed to rabbit antifibronectin IgG and finally to sheep anti-rabbit IgG conjugated microspheres. As expected, the basal lamina of surface and neural ectoderm as well as the remaining fibrous ECM were heavily decorated with microspheres, whereas control embryos treated with preimmune serum were beadless. Fibronectin was localized on the cell soma and processes of primary mesenchyme as early as stage 3. In addition, it was possible to decorate to various extents, populations of prosencephalic, mesencephalic, and rhombencephalic cranial neural crest cells. Our studies suggest that fibronectin is present in the cranium of chick embryos at earlier times than heretofore realized, and that fibronectin accumulates in a cranial to caudal gradient that reflects the sequential differentiation of the embryonic axis.

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