Ultrastructural localization of viral antigens using the unlabeled antibody-enzyme method.

1976 ◽  
Vol 24 (3) ◽  
pp. 517-526 ◽  
Author(s):  
G Wendelschafer-Crabb ◽  
S L Erlandsen ◽  
D H Walker
Keyword(s):  
Staining Method ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Model System ◽  
Shigella Dysenteriae ◽  
Specific Staining ◽  
Bacteriophage P1 ◽  
Viral Antigens ◽  
Enzyme Method ◽  
Unlabeled Antibody

Employing the unlabeled antibody enzyme method at the ultrastructural level, a comparison was made between preembedding staining and postembedding staining for the detection of viral antigens. The bacteriophage P1 absorbed to the surface of Shigella dysenteriae was used as a model system. Preembedding staining resulted in the specific deposition of peroxidase-antiperoxidase (PAP) complexes as an electron-dense coating around the viral heads. Disadvantages of the preembedding staining method included the agglutination of cells by the primary antiserum which produced a gradient of specific staining and the "bleeding" or migration of electron dense reaction product away from the sites of attached PAP complexes. The postembedding staining method had distinct advantages over the preembedding staining in that PAP complexes were deposited directly over exposed viral heads within the thin section. In addition, the specific immunostaining of viruses was uniform through the section and no artifactual migration of reaction product was observed.

Ultrastructural localization of neuropeptides in the rat brain

1978 ◽  
Vol 36 (2) ◽  
pp. 612-613
Author(s):  
F.W. Van Leeuwen
Keyword(s):  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Serial Sections ◽  
Microscopical Level ◽  
Electron Microscopical Level ◽  
Enzyme Method ◽  
Perfusion Fixation ◽  
Electron Microscopical ◽  
Unlabeled Antibody ◽  
Peroxidase Conjugate

In order to obtain specific and optimal ultrastructural localization of vasopressin and oxytocin in the hypothalamo-neurohypophyseal system of the rat, 2 staining procedures and several tissue treatments were evaluated using neurohypophyseal tissue. It appeared from these studies that post-embedding staining with the unlabeled antibody enzyme method developed by Sternberger allows greater dilution of the first antibody (anti-vasopressin, 1:4800) than the indirect procedure (1:320) using a peroxidase conjugate as second antibody. Immersion fixation with 4% formalin during 24 hours gave better results (general ultrastructure, immunoreactivity) than those obtained by perfusion fixation with 2.5% glutaraldehyde-1% paraformaldehyde or freeze substitution.Since no reliable specificity tests were performed at the electron microscopical level, tests were developed for antibodies against both vasopressin and oxytocin. For anti-vasopressin plasma neural lobes of homozygous Brattleboro rats, that are lacking vasopressin by a genet- ical defect, were used. For antibodies against both hormones serial sections were used that were alternately incubated with the antibodies.

scholarly journals THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOCYTOCHEMISTRY QUANTITATIVE COMPARISON OF SENSITIVITIES WITH AND WITHOUT PEROXIDASE-ANTIPEROXIDASE COMPLEX

1974 ◽  
Vol 22 (8) ◽  
pp. 782-801 ◽  
Author(s):  
JOHN P. PETRALI ◽  
DENNIS M. HINTON ◽  
GWEN C. MORIARTY ◽  
LUDWIG A. STERNBERGER
Keyword(s):  
Secretory Granules ◽  
Enzyme Reaction ◽  
Fold Increase ◽  
Immunocytochemical Staining ◽  
Intermediate Lobe ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Specific Staining ◽  
Enzyme Method ◽  
Unlabeled Antibody

Araldite sections of rat pituitary intermediate lobe were used with anti-17-39 adrenocorticotropin in the unlabeled antibody enzyme method to compare electron microscopic immunocytochemical staining by peroxidase-antiperoxidase complex (PAP) with antiserum or purified antibody to peroxidase followed by peroxidase (PO). Quantitation of normalized optical densities of secretory granules offered high significance comparison (P < 0.0001) of experimental with control values and of experimental values with each other. Use of purified antibody (prepared by a new density gradient method) yielded four times higher sensitivity than antiserum to PO, while a 6.5-fold increase would have been expected from the degree of contamination of anti-PO in the serum by nonanti-PO immunoglobulin. Use of PAP was four to five times more sensitive than purified anti-PO and 20 times more sensitive than antiserum to PO. The increased sensitivity of PAP is explained by the high over-all binding affinity of PO for anti-PO in the cyclic PAP molecule, thus preventing the losses of PO that occur during washing when anti-PO and PO have been applied in sequence. Identification of the characteristic, cyclic PAP molecules affords confirmation of specific staining at high resolution. In the sequential application of anti-PO and PO, no PAP molecules are formed, thus making distinction of specific from nonspecific deposition of enzyme reaction products ambiguous. With the use of anti-17-39 ACTH and the intermediate lobe, the unlabeled antibody enzyme method was 16,000-100,000 times more sensitive than radioimmunoassay.

scholarly journals ULTRASTRUCTURAL IMMUNOCYTOCHEMICAL LOCALIZATION OF LYSOZYME IN THE PANETH CELLS OF MAN

1974 ◽  
Vol 22 (6) ◽  
pp. 401-413 ◽  
Author(s):  
STANLEY L. ERLANDSEN ◽  
JONATHAN A. PARSONS ◽  
THOMAS D. TAYLOR
Keyword(s):  
Paneth Cell ◽  
Ultrastructural Level ◽  
Uranyl Acetate ◽  
Paneth Cells ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Specific Staining ◽  
Secretion Granules ◽  
Normal Sheep Serum ◽  
Unlabeled Antibody

Human lysozyme was localized immunocytochemically at the ultrastructural level within Paneth cells of man by use of the unlabeled antibody enzyme method. Specific staining for lysozyme was observed over secretion granules in the apical cytoplasm, within the region of the Golgi apparatus and within some, but not all, lysosomes. No staining was observed within the cisternae of the rough endoplasmic reticulum or other cellular organelles. In control experiments comparing semiadjacent sections of the same Paneth cell, substitution of either normal rabbit serum for rabbit antihuman lysozyme antiserum (specificity control) or normal sheep serum for sheep antirabbit immunoglobulin G antiserum (method control) completely eliminated specific staining for lysozyme. The intensity of staining for lysozyme was related to both the titer and length of exposure to antilysozyme antiserum. Specific staining was obtained in tissue embedded in Araldite or Epon and was facilitated by etching with hydrogen peroxide. No staining was observed after prolonged fixation in glutaraldehyde or treatment with uranyl acetate in block.

Combined application of monoclonal antibody to human la antigens and avidinblotin-peroxidase staining method in immunoelectron microscopy

1984 ◽  
Vol 42 ◽  
pp. 258-259
Author(s):  
K.C. Feng-Chen ◽  
B.F. Chen ◽  
A.K. Ng
Keyword(s):  
Monoclonal Antibody ◽  
Immunoelectron Microscopy ◽  
Staining Method ◽  
Ultrastructural Level ◽  
Combined Application ◽  
Antibody Method ◽  
Tissue Antigens ◽  
Pap Method ◽  
Abc Method ◽  
Unlabeled Antibody

Immunological detection of cellular and tissue antigens have been based on the widely used original enzyme labeled antibody method or the unlabeled antibody, peroxidase-anti-peroxidase (PAP) method. Recently, an improved immunoenzymatic technique for light microscopy using the avidin-biotinperoxidase complex (ABC) system was developed for cell and tissue staining, and found to be superior to the PAP technique in terms of sensitivity and specificity. The application of the ABC method in immunoelectron microscopy (IEM), however, has not been extensively evaluated. This study demonstrates that the ABC method is suitable for localizing specific cellular antigen at the ultrastructural level with monoclonal antibody (MAb), the production of which by somatic cell hybridization is now a well established procedure.

scholarly journals Quick-freezing and freeze-drying in preparation for high quality morphology and immunocytochemistry at the ultrastructural level: application to pancreatic beta cell.

1982 ◽  
Vol 30 (2) ◽  
pp. 129-138 ◽  
Author(s):  
R W Dudek ◽  
G V Childs ◽  
A F Boyne
Keyword(s):  
Beta Cell ◽  
Secretory Granules ◽  
Pancreatic Beta Cell ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Freeze Dried ◽  
Quick Freezing ◽  
Degree Of Confidence ◽  
Freeze Fixation ◽  
Unlabeled Antibody

Quick-freeze fixation and freeze-dry methods were used successfully to obtain ultrastructural localization of insulin in the pancreatic beta cell by the unlabeled antibody-enzyme technique. In unosmicated freeze-fixed and freeze-dried islets, insulin was specifically demonstrated over the dense core of the secretory granules. In osmicated freeze-fixed and freeze-dried islets, insulin antigenicity withstood the osmium tetroxide vapor treatment. In addition, the surrounding ultrastructural resolution of morphologic features was significantly improved, which allowed insulin to be localized not only in secretory granules, but also in intracellular membranous compartments, with a degree of confidence not heretofore possible. Extracellular sites of insulin positivity in the islet were also localized and possible exocytotic activity for showing insulin release was observed.

Ultrastructural localization of cellulase in Trichoderma reesei using immunocytochemistry and enzyme cytochemistry.

1983 ◽  
Vol 31 (12) ◽  
pp. 1363-1366 ◽  
Author(s):  
C M Chapman ◽  
J R Loewenberg ◽  
M J Schaller ◽  
J E Piechura
Keyword(s):  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Trichoderma Reesei ◽  
Growth Medium ◽  
Culture Medium ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Enzyme Cytochemistry ◽  
Specific Staining ◽  
Cellulase Complex

Two components of the cellulase complex (E.C. 3.2.1.4) of the fungus Trichoderma reesei were localized at the ultrastructural level. Immunocytochemistry and enzyme cytochemistry demonstrated that cellobiohydrolase and beta-1,4 glucanase were localized within cisternae of endoplasmic reticulum and within membrane complexes of cellulose-grown hyphae. Both enzymes were also present in the culture medium. Glucose-grown control hyphae lacked enzyme-specific staining, and no enzyme activity was detected in the growth medium.

Quantitative Immunocytochemistry of Corticotropin by the Unlabeled Antibody Enzyme Method with and without Peroxidase-Antiperoxidase Complex (PAP)

1973 ◽  
Vol 31 ◽  
pp. 688-689
Author(s):  
John P. Petrali ◽  
Gwen C. Moriarty ◽  
Ludwig A. Sternberger
Keyword(s):  
Hydrogen Peroxide ◽  
High Resolution ◽  
Specific Antiserum ◽  
Specific Staining ◽  
Primary Antiserum ◽  
Quantitative Immunocytochemistry ◽  
Rat Pituitary ◽  
Enzyme Method ◽  
Unlabeled Antibody

The unlabeled antibody enzyme method localizes antigen by reaction with (1) specific antiserum (primary antibodies), (2) antiserum to the primary immunoglobulin, (3) antiperoxidase (anti-PO) and (4) peroxidase (P0). The reaction is developed with hydrogen peroxide as substrate for P0 and diaminobenzidine (DAB) as co-substrate, followed by osmication. Anti-PO can be used in the form of antiserum (procedure A), purified antibody (procedure B), or steps (3) and (4) can be combined by the use of purified, soluble PAP (procedure C). Only at relatively low resolutions does identification of antigen depend on specific staining. High resolution localization depends on identification of the characteristic ring-shaped PAP molecules (Fig. 1).We have compared the sensitivities of procedures A, B and C upon staining serial Araldite sections of rat pituitary intermediate lobes using anti17-39 corticotropin as primary antiserum. PAP contained 3.33 mg anti-PO, 1.2 mg PO/ml and was essentially pure. It was employed at a standard dilution of 1:50. Antiserum and purified antibody to PO were used at standard dilutions containing equivalent amounts of anti-PO (1:24 for antiserum, 1:5 for purified antibody).

Ultrastructural Localization of Antigens by Capillary Action Immunocytochemistry

1996 ◽  
Vol 54 ◽  
pp. 40-41
Author(s):  
László G. Kömüves
Keyword(s):  
San Francisco ◽  
Colloidal Gold ◽  
Specific Binding ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Uranyl Acetate ◽  
Adhesive Tape ◽  
Distilled Water ◽  
Capillary Action ◽  
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.

scholarly journals Immunoelectron microscopic localization of herpes simplex virus antigens in infected cells using the unlabeled antibody-enzyme method.

1979 ◽  
Vol 27 (11) ◽  
pp. 1455-1461 ◽  
Author(s):  
B L Hansen ◽  
G N Hansen ◽  
B F Vestergaard
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Reaction Products ◽  
Viral Antigens ◽  
Infected Cells ◽  
Viral Morphogenesis ◽  
Simplex Virus ◽  
Unlabeled Antibody ◽  
Hsv 1

Subcellular localization of viral antigens was demonstrated during viral morphogenesis using herpes simplex virus type 1 (HSV-1) infected monolayers of rabbit cornea cells. The localization was done by immunoelectron microscopy employing the peroxidase-antiperoxidase (PAP) immunocytochemical technique and the postembedding staining method. The localization of viral antigens was followed at time intervals during infection from 2 to 19 hr. After exposure of sections to either polyspecific antibodies against total HSV-1 antigens or monospecific antibodies against HSV-1 antigen No. 8, specific immunological reaction products were identified both in the cytoplasm and nucleus after 2 hr. The distribution and quantity of reaction products varied in the infected cells during the viral morphogenesis. The present results on the subcellular distribution of the HSV-1 antigens are related to current biochemical findings.

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