scholarly journals The unlabeled antibody enzyme method. Attempted use of peroxidase-conjugated antigen as the third layer in the technique.

1977 ◽  
Vol 25 (9) ◽  
pp. 1036-1042 ◽  
Author(s):  
L A Sternberger ◽  
J P Petrali
Keyword(s):  
The Third ◽  
Enzyme Method ◽  
Soluble Peroxidase ◽  
Low Sensitivity ◽  
Antigenic Reactivity ◽  
Unlabeled Antibody ◽  
Covalently Bound

The use of covalently bound peroxidase-immunoglobulin conjugate has been compared with soluble peroxidase-antiperoxidase complex as antigen in the third layer of the unlabeled antibody enzyme method. The conjugate conferred no staining. The low sensitivity of the conjugate appeared to be due in part to interference by unconjugated immunoglobulin and in part to diminution of antigenic reactivity of immunoglobulin as a result of conjugation. The triple specificity amplification inherent in the unlabeled antibody enzyme method with peroxidase-antiperoxidase complex may be lost if a peroxidase-immunoglobulin conjugate could be prepared in such a manner that staining occurs.

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 ELECTRON MICROSCOPIC STUDY OF THE ADRENOCORTICOTROPIN-PRODUCING CELL WITH THE USE OF UNLABELED ANTIBODY AND THE SOLUBLE PEROXIDASE-ANTIPEROXIDASE COMPLEX

1972 ◽  
Vol 20 (8) ◽  
pp. 590-603 ◽  
Author(s):  
G. C. MORIARTY ◽  
N. S. HALMI
Keyword(s):  
Secretory Granules ◽  
Staining Intensity ◽  
Electron Microscopic ◽  
Early Effect ◽  
Acth Cell ◽  
Maximal Diameter ◽  
Soluble Peroxidase ◽  
Ultrathin Sections ◽  
Acth Secreting ◽  
Unlabeled Antibody

The technique involving use of unlabeled antibody and the peroxidase-antiperoxidase complex was used to identify the adrenocorticotropin (ACTH)-secreting cell in the anterior pituitary lobe of the rat and to localize ACTH in it electron microscopically in ultrathin sections. The ACTH cell is star-shaped, with processes extending around other cells, and contains secretory granules of a maximal diameter of 300 mµ arranged peripherally along the plasma membrane. Stain was observed on secretory granules, around them, in the Golgi complex and in rough endoplasmic reticulum. One day after adrenalectomy, the ACTH cell is degranulated and the staining intensity of its remaining granules and cytoplasm is decreased, suggesting release of ACTH stores. If cortisol is given 6 hr after adrenalectomy, 18 hr later the ACTH cells are well granulated and the granules stain more intensely than normal. In addition, staining around the granules and throughout the cytoplasm is more intense, suggesting that an early effect of cortisol is to block release of ACTH. Twenty-one days after adrenalectomy, the ACTH cells are greatly increased in numbers and have complex, tortuous processes filled with intensely stained secretory granules.

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.

Radiochemical Analysis of Orthophosphate Concentrations and Seasonal Changes in the Flux of Orthophosphate to Seston in Two Canadian Shield Lakes

1980 ◽  
Vol 37 (3) ◽  
pp. 479-487 ◽  
Author(s):  
S. N. Levine ◽  
D. W. Schindler
Keyword(s):  
Seasonal Changes ◽  
Phosphate Concentration ◽  
Radiochemical Analysis ◽  
Small Lakes ◽  
Isotopic Equilibrium ◽  
Experimental Lakes Area ◽  
The Third ◽  
Long Period ◽  
Low Sensitivity ◽  
Shield Lakes

Seasonal changes in the concentration and dynamics of phosphate–phosphorus were studied in two small lakes, one oligotrophic and one artificially eutrophied. Because the molybdate blue phosphate technique frequently overestimates phosphate concentrations, three radiochemical assays were used. One, involving sephadex fractionation, was unsatisfactory because of the long period required for high molecular weight phosphorus fractions to reach isotopic equilibrium. The second method was unusable both for epilimnion waters within the Experimental Lakes Area, because of its low sensitivity, and for hypolimnion waters, due to interference from nonphosphate compounds. The third method, Rigler's bioassay, indicated that PO4-P in both lakes seldom exceeded 0.1 μg∙L−1, even under anoxic conditions. Organisms, and not mineral reactions, appeared to regulate the phosphate concentration at all depths in the lakes.Key words: phosphorus dynamics, orthophosphate

scholarly journals Immunocytochemistry with osmium-fixed tissue. I. Light microscopic localization of growth hormone and prolactin with the unlabeled antibody-enzyme method.

1979 ◽  
Vol 27 (4) ◽  
pp. 867-872 ◽  
Author(s):  
D G Baskin ◽  
S L Erlandsen ◽  
J A Parsons
Keyword(s):  
Growth Hormone ◽  
Epoxy Resin ◽  
Alcoholic Solution ◽  
Immunocytochemical Staining ◽  
Antigenic Determinants ◽  
Fixed Tissue ◽  
Morphological Evaluation ◽  
Enzyme Method ◽  
Thin Plastic ◽  
Unlabeled Antibody

Growth hormone and prolactin were localized on thin plastic sections of rat anterior pituitary gland and mammosomatotropic tumor MtTW15 that were fixed with osmium tetroxide (alone,mixed with aldehydes, or after aldehydes). Intense immunocytochemical staining for both antigens was obtained after plastic was removed from sections with an alcoholic solution of sodium hydroxide. The results indicated that antigenic determinants of rat prolactin and growth hormone were not completely destroyed or inactivated by fixation with osmium and embedment in epoxy resin, and that removal of the polymerized epoxy resin was necessary to obtain light microscopic postembedding immunocytochemical staining of these antigens. The results also demonstrated that tissues which have been conventionally processed for morphological evaluation by electron microscopy may be suitable for postembedding immunocytochemical staining of some antigens for light microscopy.

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