The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

1985 ◽  
Vol 43 ◽  
pp. 426-429
Author(s):  
George H. Herbener ◽  
Antonio Nanci ◽  
Moise Bendayan
Keyword(s):  
Tissue Section ◽  
Colloidal Gold ◽  
Unit Area ◽  
Protein A ◽  
Extracellular Proteins ◽  
Gold Complex ◽  
Second Step ◽  
Igg Antibodies ◽  
Tissue Sections ◽  
Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

scholarly journals Double immunocytochemical labeling applying the protein A-gold technique.

1982 ◽  
Vol 30 (1) ◽  
pp. 81-85 ◽  
Author(s):  
M Bendayan
Keyword(s):  
Tissue Section ◽  
Protein A ◽  
Antigenic Site ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Proteins ◽  
Zymogen Granules ◽  
Double Labeling ◽  
Gold Particles ◽  
Antigenic Sites

In the present study we report the modifications and the different steps of the protein A-gold (pAg) technique that allow the simultaneous demonstration of two antigenic sites on the same tissue section. The labeling is carried out in the following manner: face A of the tissue section is incubated with an antiserum followed by a pAg complex prepared with large gold particles; face B of the same tissue section is then incubated with a second antiserum followed by a pAg complex prepared with small gold particles. Each of the pAg complexes reveals a different antigenic site on opposite faces of the tissue section. The transparency of the section in the electron beam allows the visualization of the gold particles present on both faces. The double labeling pAg technique was applied for the simultaneous demonstration of two secretory proteins in the same Golgi, condensing vacuoles, and zymogen granules of the rat pancreatic acinar cells.

scholarly journals Use of the protein A-gold technique for the morphological study of vascular permeability.

1980 ◽  
Vol 28 (11) ◽  
pp. 1251-1254 ◽  
Author(s):  
M Bendayan
Keyword(s):  
Endothelial Cells ◽  
Vascular Permeability ◽  
Ultrastructural Study ◽  
Morphological Study ◽  
Protein A ◽  
Gold Complex ◽  
Buffer Solution ◽  
General Applicability ◽  
Immunocytochemical Technique ◽  
Tissue Sections

The protein A-gold immunocytochemical technique has been applied for the ultrastructural study of vascular permeability. A capillary preparation was perfused for 30 min with a buffer solution containing albumin. After fixation and embedding, the albumin molecules were revealed on the tissue sections using anti-albumin antiserum and the protein A-gold complex. A specific labeling was obtained over the capillary lumen, the vesicular structures of the endothelial cells, and the intercapillary space. These results allowed us to conclude that the protein A-gold technique is suitable and of general applicability for the morphological study of vascular permeability.

scholarly journals Applications of immunocolloids in light microscopy. IV. Use of photochemical silver staining in a simple and efficient double-staining technique.

1985 ◽  
Vol 33 (12) ◽  
pp. 1247-1251 ◽  
Author(s):  
C Manigley ◽  
J Roth
Keyword(s):  
Light Microscopy ◽  
Silver Staining ◽  
Colloidal Gold ◽  
Protein A ◽  
Staining Technique ◽  
Gold Complex ◽  
Double Staining ◽  
Red Color ◽  
Gold Immunolocalization ◽  
Red Coloration

We report the development of a new light-microscopic double-staining technique using colloidal gold as sole marker. The contrasting color to the red of colloidal gold is achieved by the application of photochemical silver reaction. The silver reaction, which is principally performed at the end of the first staining sequence, converts the red color of a gold-labeled reagent into black. This contrasts clearly with the red coloration that results from the second incubation sequence without silver reaction. For antigen double staining, the same protein A-gold complex can be used to provide the black and the red color, thus rendering the technique very economical. Alternatively, combination of protein A-gold immunolocalization and lectin-gold staining is possible, as is combined lectin-gold staining.

Ultrastructural localization of serotonin and polypeptide YY (PYY) in endocrine cells of the human rectum.

1986 ◽  
Vol 34 (6) ◽  
pp. 719-726 ◽  
Author(s):  
A I Lukinius ◽  
J L Ericsson ◽  
M K Lundqvist ◽  
E M Wilander
Keyword(s):  
Colloidal Gold ◽  
Endocrine Cells ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Enterochromaffin Cells ◽  
Cell Type ◽  
Low Concentration ◽  
Antigenic Sites ◽  
Human Rectum ◽  
Ultrathin Sections

This study was performed with the aim of ultrastructurally localizing serotonin and polypeptide YY (PYY) in the endocrine cells of the human rectum. Existing basic methods for immunolocalization of antigenic sites in ultrathin sections were tested and modified to allow reproducible results with distinct localization of marker (colloidal gold probes coupled either to IgG or protein A). Probes signifying presence of serotonin were distinctly localized over all heteromorphous granules in argentaffin cells and, in addition, over some of the more monomorphous, rounded granules in a second cell type whose granules all were covered by probes showing localization of the PYY antigen. The results suggest that serotonin in endocrine cells of the gut is not confined to the enterochromaffin type but may also be present in trace amounts in non-enterochromaffin endocrine cells storing peptide hormones. Since probes marking sites of PYY were deposited over some heteromorphous granules in enterochromaffin cells, the evidence obtained also suggests that PYY may occur in low concentration in these cells. The distribution of probes in the sections indicated that antigenic sites were confined to granules in the cells.

Characterization of an optimal protein A-gold complex: Improvement of the labeling efficiency

1990 ◽  
Vol 48 (3) ◽  
pp. 942-943
Author(s):  
Lucian Ghitescu ◽  
Moise Bendayan
Keyword(s):  
Polyethylene Glycol ◽  
Gold Particle ◽  
Colloidal Gold ◽  
Specific Activity ◽  
Protein A ◽  
Gold Complex ◽  
Dense Layer ◽  
Gold Particles ◽  
Optical Absorbance

By using 125I protein A, we have prepared complexes of this immunoprobe with colloidal gold particles having diameters ranging from 5 to 15 nm. Various concentrations of protein A (from 35 to 1000 nM) of known specific activity were employed in order to obtain a spectrum of conjugates differing not only in sizes, but also in amounts of protein A they carry. After ultracentrifugation through a step gradient (15, 30 and 70% w/v) of sucrose, the protein A-gold complexes were quantitatively recovered from the most dense layer, and diluted in a 0.01 M phosphate buffer containing 0.01% polyethylene glycol. The conjugates did not contain free protein A, as proven by the lack of radioactivity in the intermediate sucrose layers. By correlating the radioactivity of the complexes with their particle densities (inferred from the optical absorbance at 520 nm), the number of protein A molecules bound per gold particle was calculated for each experimental condition.

scholarly journals Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections.

1982 ◽  
Vol 30 (7) ◽  
pp. 691-696 ◽  
Author(s):  
J Roth
Keyword(s):  
Protein A ◽  
Second Step ◽  
Staphylococcal Protein A ◽  
Paraffin Sections ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Antigen Localization ◽  
Color Mixing ◽  
Electron Microscopic Localization

The protein A-gold (pAg) complex, a useful reagent for electron microscopic localization of antigens in thin sections, is tested for its suitability as second step reagent in light microscopic immunohistochemistry. In addition, the preparation of colloidal silver, its complex formation with staphylococcal protein A and the application of the protein A-silver complex for antigen localization in paraffin sections is reported. The antigens were visualized in a two-step technique with specific antisera in the first incubation step and pAg or pA-silver as a general second step reagent. The pAg complex gives a red coloration of antigenic sites, whereas the pA-silver stained yellow. The contrasting color provided by the two immunocolloids allowed localization of two antigens in the same section. No color mixing occurred, showing that removal of the antibodies of the first staining sequence is unnecessary. Staining is virtually permanent with the light microscopic immunocolloid method. It is concluded that pAg and pA-silver complexes are useful as general second step reagents for the localization of a variety of antigens in paraffin sections.

scholarly journals Protein AG-gold complex: an alternative probe in immunocytochemistry.

1991 ◽  
Vol 39 (8) ◽  
pp. 1057-1065 ◽  
Author(s):  
L Ghitescu ◽  
Z Galis ◽  
M Bendayan
Keyword(s):  
Binding Sites ◽  
Colloidal Gold ◽  
Protein A ◽  
Particle Surface ◽  
Surface Protein ◽  
Immunoblot Analysis ◽  
Gold Complex ◽  
Protein G ◽  
Staphylococcal Protein A ◽  
Monoclonal Immunoglobulins

The purpose of this study was to evaluate the use of protein AG tagged with colloidal gold as a reliable immunocytochemical reagent. Protein AG is a recombinant of 47.3 KD molecular weight and pI = 4.3, which displays immunoglobulin Fc binding sites for both staphylococcal protein A and streptococcal protein G. It adsorbs to 10-nm colloidal gold particles with a lower affinity than does protein A, and is saturable. A maximal number of 12 protein AG molecules could be accommodated on the gold particle surface. Protein AG-gold conjugates yielded positive signals in post-embedding immunocytochemical assays when used as a secondary reagent in conjunction with several species and classes of polyclonal (rabbit, goat, sheep, guinea pig) and mouse monoclonal immunoglobulins (IgG1, IgG2, and IgG3). In addition, protein AG-gold was found to be a useful reagent in immunoblot analysis because of its ability to bind and identify nitrocellulose-immobilized IgGs (rabbit, mouse, goat, sheep, rat, and cow). Its spectrum of specificity towards various types of antibodies combines those of the parental protein A and protein G molecules. The protein AG-gold complex therefore appears to be a highly versatile and convenient alternative probe for immunochemical and immunocytochemical studies.

Immuno-gold post-embedding labeling for electron microscopy: Technical procedures and potential limitations

1990 ◽  
Vol 48 (3) ◽  
pp. 142-143
Author(s):  
Moise Bendayan
Keyword(s):  
Protein A ◽  
Cellular Structures ◽  
Protein G ◽  
Secondary Antibody ◽  
Antibody Molecule ◽  
Gold Particles ◽  
Tissue Sections ◽  
Antigenic Sites

The successful post-embedding immunolabeling of tissue sections requires several essential steps.1) The use of an highly specific and well characterized antibody.2) The use of an efficient probe that will bind the antibody molecule.3) The use of tissue sections prepared for microscopy which will combine good preservation of the cellular structures and retention of protein antigenicity.The characterization of the antibodies and their monospecificity are prerequisite conditions that will determine the final quality of the results.Several immunoprobes are available for the labeling of antigenic sites. The use of protein A or protein G, secondary antibody molecules tagged with colloidal gold particles or the biotin-avidin gold system, have been able to generate very good and reliable results of similar value. The question on the size of the gold particles remains a decision that should be taken according to the results expected. Labeling of large cellular structures and their visualization at low magnification require relatively large gold particles (10 to 15 nm).

scholarly journals Novel secretory granule morphology in physically fixed pancreatic islets.

1984 ◽  
Vol 32 (9) ◽  
pp. 929-934 ◽  
Author(s):  
R W Dudek ◽  
A F Boyne ◽  
T M Charles
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Gold Complex ◽  
Core State ◽  
Dense Cores ◽  
Antigenic Sites ◽  
Granule Morphology ◽  
Rat Islets Of Langerhans ◽  
Filamentous Material ◽  
Kinetics Of

Protein A-gold immunocytochemistry has been applied to physically fixed beta cells from rat islets of Langerhans. The punctate nature of the gold particles permits improved resolution of the antigenic sites without obscuring the fine ultrastructural preservation obtained by physical fixation. There is a filamentous material within the halo of the secretory granules that is not preserved by aqueous, chemical fixation. When viewed in stereo the filaments appear as an annular cobweb or a series of wheel spokes attached to a centrally located hub (the dense core of the granule). The filaments demonstrate insulin-like immunoreactivity using the protein A-gold technique. The immunoreactivity appears to be restricted to the filaments and the surface of the dense cores. This may be a consequence of the preservation of a solid, insolubilized core state that resists penetration by the antibody and/or the protein A-gold complex. However, the evidence that there is a halo pool of insulin which is separate from the massive core aggregate suggests that i) correspondingly massive exocytotic pits may not be as mandatory for insulin release as has been assumed and ii) the complex kinetics of insulin secretion may be, in part, a reflection of multiple insulin compartments within secretory granules.

scholarly journals Immunohistochemical Detection of Porcine Rotavirus Using Immunogold Silver Staining (IGSS)

1992 ◽  
Vol 4 (1) ◽  
pp. 3-7 ◽  
Author(s):  
Ronald Magar ◽  
Renée Larochelle
Keyword(s):  
Silver Staining ◽  
Cultured Cells ◽  
Protein A ◽  
Gold Complex ◽  
Immunogold Electron Microscopy ◽  
Group A Rotavirus ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin Embedded ◽  
Group A ◽  
Immune Aggregates

Immunogold silver staining (IGSS) was applied for the detection of porcine group A rotavirus in formalin-fixed paraffin-embedded tissue sections of small intestine. Prior to the application of IGSS, the reactivity of protein A-gold as a marker was tested with group-specific antiserum in immunogold electron microscopy. Immune aggregates were intensely and specifically labeled with the gold complex. Application of IGSS to tissue sections resulted in specific dark staining of villous enterocytes infected by group A rotavirus. This method also proved effective for the detection of rotaviral antigen in infected cultured cells. The IGSS method may be suitable for routine diagnostic detection of rotaviral infections and may have application for detection of other viral pathogens of veterinary importance.

Sign in / Sign up

Export Citation Format

Share Document