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 Ultrastructural localization of intracellular antigens by the use of protein A-gold complex.

1978 ◽  
Vol 26 (12) ◽  
pp. 1074-1081 ◽  
Author(s):  
J Roth ◽  
M Bendayan ◽  
L Orci
Keyword(s):  
Endocrine Pancreas ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Gold Complex ◽  
Secretory Proteins ◽  
Staphylococcal Protein A ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Step Procedure ◽  
General Method

An immunocytochemical technique for the demonstration of intracellular antigens (secretory proteins) on thin sections is reported. Staphylococcal protein A which reacts with the Fc fragment of IgG molecules was labeled with colloidal gold as a marker. The antigenic sites were visualized on aldehyde-fixed and Epon-embedded tissue in a two step procedure. The specific antisera were applied to thin sections for binding to the antigens and then visualized by the protein A-gold complex. By using this technique different secretory proteins of the exocrine and endocrine pancreas were localized. The protein A-gold technique is proposed as a general method for visualization of antigenic sites on thin sections.

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 Immunocytochemical localization of D-amino acid oxidase in the central clear matrix of rat kidney peroxisomes.

1986 ◽  
Vol 34 (12) ◽  
pp. 1709-1718 ◽  
Author(s):  
N Usuda ◽  
S Yokota ◽  
T Hashimoto ◽  
T Nagata
Keyword(s):  
Amino Acid ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
Thin Sections ◽  
Gold Particles

Light and electron microscopic localizations of D-amino acid oxidase (DAO) in rat kidney was investigated using immunoenzyme and protein A-gold techniques. The enzyme was purified from rat kidney homogenate and its antibody was raised in rabbits. By Ouchterlony double-diffusion analysis and immunoblot analysis with anti-(rat kidney DAO) immunoglobulin, the antibody was confirmed to be monospecific. The tissue sections (200 micron thick) of fixed rat kidney were embedded in Epon or Lowicryl K4M. Semi-thin sections were stained for DAO by the immunoenzyme technique after removal of epoxy resin for LM, and ultra-thin sections of Lowicryl-embedded material were labeled for DAO by the protein A-gold technique for EM. By LM, fine cytoplasmic granules of proximal tubule were stained exclusively. Among three segments of proximal tubules, and S2 and S3 segments were heavily stained but the S1 segment only weakly so. By EM, gold particles indicating the antigenic sites for DAO were exclusively confined to peroxisomes. Within peroxisomes, the gold particles were localized in the central clear matrix but not in the peripheral tubular substructures. The results indicate that D-amino acid oxidase in rat kidney is present exclusively in peroxisomes in the proximal tubule and that within peroxisomes it is found only in central clear matrix and not in the peripheral tubular substructures.

scholarly journals Immunocytochemical localization of Na+,K+-ATPase catalytic polypeptide in mouse choroid plexus.

1986 ◽  
Vol 34 (2) ◽  
pp. 189-195 ◽  
Author(s):  
S A Ernst ◽  
J R Palacios ◽  
G J Siegel
Keyword(s):  
Choroid Plexus ◽  
Epithelial Transport ◽  
Protein A ◽  
Fluid Secretion ◽  
Physiological Data ◽  
Electron Microscopic ◽  
Antibody Technique ◽  
Immunogold Staining ◽  
Thin Sections ◽  
Membrane Surfaces

Na+,K+-ATPase plays a central role in the mechanism of cerebrospinal fluid secretion by the choroid plexus. We have used an antiserum to the 100 KD catalytic polypeptide of the enzyme purified from mouse brain (30) to localize the catalytic unit in mouse choroid plexus at the light and electron microscopic levels. Pre-embedding immunostaining with the peroxidase-conjugated second antibody technique showed that microvillar borders facing the ventricle were intensely reactive. In contrast, basal and lateral plasma membrane surfaces were devoid of activity. Identical localization was obtained with a post-embedding procedure in which protein A-gold was used to stain immunoreactive sites on thin sections of Lowicryl-embedded tissue. For comparison, immunogold staining was shown to be restricted to basolateral membranes of kidney medullary ascending thick limbs. The apical localization of Na+,K+-ATPase in choroid plexus is in striking contrast to the almost exclusive basolateral localization seen in other ion-transporting tissues. The immunocytochemical data are completely consistent with physiological data on choroidal epithelial transport and with light microscopic autoradiographic localization of [3H]-ouabain binding sites.

scholarly journals Electron microscopic localization of chromogranin A in osmium-fixed neuroendocrine cells with a protein A-gold technique.

1987 ◽  
Vol 35 (7) ◽  
pp. 795-801 ◽  
Author(s):  
S A Hearn
Keyword(s):  
Neuroendocrine Tumors ◽  
Chromogranin A ◽  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Protein A ◽  
Neuroendocrine Cells ◽  
Neurosecretory Granules ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Carotid Body Tumors

An antibody (LK2H10) to chromogranin A has been recommended for use in ultrastructural identification of neuroendocrine secretory granules. Previous studies have demonstrated immunoreactive chromogranin A in specimens prepared for electron microscopy by glutaraldehyde fixation only. In this study, the effect of specimen post-fixation by osmium tetroxide on post-embedding localization of chromogranin A was evaluated. Human tissues from benign endocrine glands, neuroendocrine tumors, and non-neuroendocrine tumors were post-fixed in osmium, embedded in epoxy resin, and the sample thin sections immunolabeled using a protein A-gold technique. Chromogranin A-positive neurosecretory granules were detected in pancreatic islets, adrenal medulla, stomach, ileum, anterior pituitary, and parathyroid. Mid-gut carcinoids, bronchial carcinoids, pheochromocytomas, paragangliomas, carotid body tumors, and thyroid medullary carcinomas contained immunoreactive granules. Cytoplasmic granules in non-neuroendocrine tumors did not react for chromogranin A. Tissues post-fixed in osmium tetroxide had optimally preserved ultrastructural features, and use of this fixative is compatible with postembedding localization of chromogranin A in neurosecretory granules.

scholarly journals Conjugation of horseradish peroxidase to staphylococcal protein A with benzoquinone, glutaraldehyde, or periodate as cross-linking reagents.

1981 ◽  
Vol 29 (2) ◽  
pp. 266-270 ◽  
Author(s):  
H Nygren ◽  
H A Hansson
Keyword(s):  
Horseradish Peroxidase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Molecular Size ◽  
High Yield ◽  
Second Step ◽  
Cross Linking ◽  
Staphylococcal Protein A ◽  
Staphylococcal Protein

Horseradish peroxidase was conjugated to Staphylococcal protein A by three different two-step procedures using an increasing excess of peroxidase in the second step reaction. The yield of conjugated protein A was analyzed by SDS-polyacrylamide gel electrophoresis. Conjugation of peroxidase to protein A with benzoquinone or glutaraldehyde as cross-linking reagents at a 3- to 4-fold molar excess of peroxidase resulted in a high yield of coupled protein A with conjugates of low molecular size. Conjugation of peroxidase to protein A by the periodate method resulted in a high yield of coupled protein A with polymeric conjugates of large molecular size. Based on these results, conjugates produced with glutaraldehyde as cross-linking reagents were further analyzed. The capacity of the conjugates to precipitate human immunoglobulin evaluated by radial immunodiffusion was found to be reduced to about 50% of that of native protein A. Conjugates produced with glutaraldehyde as cross-linking reagent retained 70% of the enzyme activity of native peroxidase.

scholarly journals A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride.

1986 ◽  
Vol 34 (11) ◽  
pp. 1449-1457 ◽  
Author(s):  
A I Levey ◽  
J P Bolam ◽  
D B Rye ◽  
A E Hallanger ◽  
R M Demuth ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Tyrosine Hydroxylase ◽  
Substance P ◽  
Light And Electron Microscopy ◽  
Immunoperoxidase Staining ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Staining Methods ◽  
Antigen Localization ◽  
Color Mixing

Very few double-antigen staining methods are available that are applicable to both light and electron microscopy. The objective of this study was to develop for localization of two neural antigens simultaneously a procedure which would be sensitive, simple to perform, offer permanent reaction products, and permit correlated light and ultrastructural analysis. The method employs sequential immunoperoxidase staining without antibody elution, in which the first sequence of antibodies is visualized with 3,3'-diaminobenzidine (DAB) and the second with benzidine dihydrochloride (BDHC). The DAB reaction product (brown and diffuse) was easily distinguishable from the BDHC deposit (blue, granular, and more electron-dense) by both light and electron microscopy. The procedure was used to simultaneously localize choline acetyltransferase-and either substance P or tyrosine hydroxylase in rat brain at both light and ultrastructural levels. Control experiments demonstrated the absence of both color mixing and antibody crossreactions, even when both primary antibodies were from the same species. This study demonstrates the usefulness of BDHC as a chromogen for immunoperoxidase staining either alone or in combination with DAB, and describes a double method which should have wide applicability for detailed studies of most pairs of antigens at both light and ultrastructural levels.

scholarly journals A rapid method for immunofluorescent staining of paraffin sections using iron-containing protein A microspheres.

1981 ◽  
Vol 29 (7) ◽  
pp. 870-873 ◽  
Author(s):  
K J Widder ◽  
A E Senyei ◽  
J L Burchette ◽  
H Ovadia ◽  
P Y Paterson
Keyword(s):  
Rapid Method ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Immunofluorescent Staining ◽  
Staphylococcal Protein A ◽  
Paraffin Sections ◽  
Formalin Fixed Paraffin ◽  
Blue Reaction ◽  
Antigen Antibody ◽  
Formalin Fixed

A method to rapidly perform immunofluorescence or light microscopic staining on formalin-fixed paraffin sections has been devised utilizing magnetic albumin microspheres containing Staphylococcal protein A. Because the protein A constituent of the microspheres has the property of binding the Fc portion of immunoglobulin G (IgG) class antibodies, the microspheres can be used to rapidly bind antigen-antibody complexes by the Fc portion of the antibody. Deparaffinized sections were stained with fluorescein isothiocyanate-conjugated antibody (IgG fractions) by standard techniques, after which the protein A microspheres were layered over the sections. Distinct fluorescence of sections was noted with the addition of the microspheres, whereas only autofluorescence was present with direct staining alone. The microspheres were also visualized by light microscopy by a subsequent Prussian blue reaction, staining the Fe3O4 within the microsphere matrix. This method represents a more rapid method for identifying antigens in tissues embedded in paraffin than has previously been reported.

scholarly journals Identification of immunoreactive sites in bovine parathyroid cells to antibodies raised against the NH2-terminal sequence of parathyroid hormone.

1979 ◽  
Vol 27 (8) ◽  
pp. 1209-1214 ◽  
Author(s):  
W Limacher ◽  
P Wild ◽  
E Manser ◽  
H Lutz
Keyword(s):  
Parathyroid Hormone ◽  
Protein A ◽  
Electron Microscopic Level ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Protein A ◽  
Terminal Sequence ◽  
Electron Microscopic ◽  
Secretion Granules ◽  
Parenchymal Cells ◽  
Bovine Parathyroid Hormone

The NH2-terminal sequence of bovine parathyroid hormone (1-84) was localized with different immunocytochemical methods on the light and electron microscopic level in bovine parathyroid glands and in isolated bovine parathyroid parenchymal cells. The peroxidase labeled staphylococcal protein A and the peroxidase anti-peroxidase method were found to be advantageous for light and electron microscopic localization, respectively. Reaction product was found light microscopically in the cytoplasma of the parenchymal cells and electron microscopically largely over the secretion granules of the parenchymal cells. The immunoreactive sites were subsequently identified to represent only intact parathyroid hormone (1-84) by gel electrophoresis derived enzyme linked immunosorbent assay.

Immuno-electron microscopic identification of Methanosarcina spp. in anaerobic digester fluid

1985 ◽  
Vol 31 (9) ◽  
pp. 839-844 ◽  
Author(s):  
Ralph W. Robinson ◽  
Gregory W. Erdos
Keyword(s):  
Protein A ◽  
Methanobacterium Thermoautotrophicum ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Methanosarcina Mazei ◽  
Pure Cultures ◽  
Electron Microscopic Identification ◽  
Mixed Samples ◽  
Heavy Labelling

Whole cell antibodies against Methanosarcina mazei strain S6 were used with protein A – colloidal gold to identify bacteria in thin sections of samples from anaerobic methane producing digesters. It was possible to identify bacteria at the genus level and to show relatedness at the species and strain levels. Heavy labelling was observed on thin sections of the immunogenic strain S6. Lighter labelling was observed with pure cultures of M. mazei strain LYC. Pure cultures of Methanococcus voltae or Methanobacterium thermoautotrophicum did not label. In samples from mesophilic sewage digesters, sarcinal colonies of bacteria similar to Methanosarcina showed heavy labelling per cell while colonies from 55 °C digesters show lighter labelling. A few free cocci, which were released from the sarcinal colonies, also labelled. Labelling was not observed on other bacterial forms in either set of digesters. These studies indicate that a collection of bacterial antibodies can be used to identify and map bacteria in situ in mixed samples.

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