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

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.

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A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

Journal of Histochemistry & Cytochemistry ◽

10.1177/23.9.1176760 ◽

1975 ◽

Vol 23 (9) ◽

pp. 666-677 ◽

Cited By ~ 83

Author(s):

S L Erlandsen ◽

J A Parsons ◽

J P Burke ◽

J A Redick ◽

D E Van Orden ◽

...

Keyword(s):

Growth Hormone ◽

Gamma Globulin ◽

Cross Reactivity ◽

Model Systems ◽

Electron Microscopic Level ◽

Immunocytochemical Staining ◽

Electron Microscopic ◽

Primary Antiserum ◽

Enzyme Method ◽

Unlabeled Antibody

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.

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Immunocytochemical localization of renin in the submandibular gland of the mouse.

Journal of Histochemistry & Cytochemistry ◽

10.1177/26.10.363930 ◽

1978 ◽

Vol 26 (10) ◽

pp. 855-861 ◽

Cited By ~ 24

Author(s):

E Gresik ◽

A Michelakis ◽

T Barka ◽

T Ross

Keyword(s):

Submandibular Gland ◽

Adult Mouse ◽

Secretory Granules ◽

Microscopic Level ◽

Electron Microscopic Level ◽

Light Microscopic Level ◽

Electron Microscopic ◽

Granular Convoluted Tubule ◽

Enzyme Method ◽

Unlabeled Antibody

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/20.8.590 ◽

1972 ◽

Vol 20 (8) ◽

pp. 590-603 ◽

Cited By ~ 102

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.

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ULTRASTRUCTURAL IMMUNOCYTOCHEMISTRY WITH UNLABELED ANTIBODIES AND THE PEROXIDASE-ANTIPEROXIDASE COMPLEX A TECHNIQUE MORE SENSITIVE THAN RADIOIMMUNOASSAY

Journal of Histochemistry & Cytochemistry ◽

10.1177/21.9.825 ◽

1973 ◽

Vol 21 (9) ◽

pp. 825-833 ◽

Cited By ~ 76

Author(s):

GWEN C. MORIARTY ◽

C. MICHAEL MORIARTY ◽

LUDWIG A. STERNBERGER

Keyword(s):

Staining Intensity ◽

Poor Quality ◽

Immunocytochemical Staining ◽

Great Promise ◽

Electron Microscopic ◽

Immunocytochemical Technique ◽

Titration Curves ◽

Rat Pituitary ◽

Normal Rat ◽

Unlabeled Antibody

Titration curves were developed with antisera to 17-39ACTH (adrenocorticotropin) and 1-39ACTH with the techniques of radioimmunoassay and electron microscopic immunocytochemistry. For the latter method, the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was used to stain normal rat pituitary intermediate lobes. By radioimmunoassay standards, the 17-39ACTH antiserum was of poor quality. Its titration curve exhibited a flat slope and it did not bind a significant amount of labeled antigen beyond a 1: 30 dilution. However, immunocytochemical staining was detected with this antiserum at dilutions as high as 1:1,500. The antiserum to 1-39ACTH was of better quality by radioimmunoassay standards. It bound 45% of the labeled antigen at a dilution of 1:5,000. Immunocytochemical staining intensity was nearly maximal at a 1:5,000 dilution and decreased progressively to a limiting value at 1:16,000. However, when incubation times in the antisera were increased from 3 min to match those of the radioimmunoassay (48 hr) maximal staining was achieved at dilutions as great as 1:512,000 where only trace amounts of the labeled antigen were bound in the radioimmunoassay. It was concluded that the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was sensitive enough to detect antibodies in sera of low titer and/or avidity which are not detected by a radioimmunoassay. The technique holds great promise for a sensitive assay system.

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/27.4.109497 ◽

1979 ◽

Vol 27 (4) ◽

pp. 867-872 ◽

Cited By ~ 69

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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Immuno electron microscopic localization of subcellular fibrillar elements using the unlabeled antibody enzyme method

Ultramicroscopy ◽

10.1016/0304-3991(79)90048-2 ◽

1979 ◽

Vol 4 (1) ◽

pp. 127-128 ◽

Cited By ~ 1

Author(s):

M. de Brabander ◽

J. de Meyt ◽

M. Joniautt ◽

G. Geuens

Keyword(s):

Electron Microscopic ◽

Microscopic Localization ◽

Enzyme Method ◽

Unlabeled Antibody ◽

Electron Microscopic Localization

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Four unlabeled antibody bridge techniques: a comparison.

Journal of Histochemistry & Cytochemistry ◽

10.1177/29.12.7033366 ◽

1981 ◽

Vol 29 (12) ◽

pp. 1397-1404 ◽

Cited By ~ 135

Author(s):

P Ordronneau ◽

P B Lindström ◽

P Petrusz

Keyword(s):

Horseradish Peroxidase ◽

Gamma Globulin ◽

Staining Intensity ◽

Reaction Products ◽

Electron Microscopic ◽

Microscopic Studies ◽

Immunocytochemical Techniques ◽

Unlabeled Antibody ◽

Shape And Size

Four unlabeled antibody immunocytochemical techniques, the "single bridge" (Avrameas S: Immunocytochemistry 6:825, 1969; Mason TE, Phifer RF, Spicer SS, Swallow RS, Dreskin RD: J Histochem Cytochem 17:190, 1969a; Sternberger LA, Cuculis JJ: 1969), the "single peroxidase-antiperoxidase (PAP)" (Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG: J Histochem Cytochem 18:315, 1970), the "double PAP" (Vacca LL, Rosario SL, Zimmerman EA, Tomashefsky P, Ng P-Y, Hsu KC: J Histochem Cytochem 23:208, 1975) and the "double bridge" (Ordronneau P, Petrusz P: Am J Anat 158:491, 1980) were compared at both the light and electron microscopic levels. The "double" procedures involved repeating incubations with the bridge antibody, in this case, sheep anti-rabbit gamma globulin, followed either by a second PAP step for the "double PAP" or a second anti-horseradish peroxidase step and a single incubation in horseradish peroxidase for the "double bridge." At both the light and electron microscopic levels the staining intensity was greater with the "double" techniques than with the "single" ones. This is probably due to amplification achieved with the second sheep anti-rabbit gamma globulin step, permitting an increase in the number of horseradish peroxidase molecules bound for each molecule of tissue-bound primary antibody. Also, the quality of the various commercial PAP preparations tested was variable. With the weaker ones the staining intensity could be increased by performing an incubation in fresh horseradish peroxidase after the PAP step. Finally, in electron microscopic studies, the reaction products formed in both the bridge and PAP procedures were identical in shape and size.

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/24.3.57192 ◽

1976 ◽

Vol 24 (3) ◽

pp. 517-526 ◽

Cited By ~ 5

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010007388x ◽

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).

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The Pituitary Response to Stress: An Immunocytochemical - Cytophysiologic Study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073775 ◽

1973 ◽

Vol 31 ◽

pp. 666-667

Author(s):

Gwen C. Moriarty ◽

C. M. Moriarty

Keyword(s):

Electron Microscopy ◽

Secretory Granules ◽

Anterior Lobe ◽

Intermediate Lobe ◽

Adrenal Cells ◽

Response To Stress ◽

Parenchymal Cells ◽

Unlabeled Antibody ◽

Adrenalectomized Rats

Physiologic studies have shown that there is a peptide in mammalian neurointermediate lobes with immunologic and biologic properties similar to adrenocorticotropin (ACTH). Immunocytochemists have localized the peptide in the secretory granules of the intermediate lobe parenchymal cells and in rostral zone anterior lobe ACTH cells. [Moriarty, G. C. and Halmi, N. S., Z. Zellforsch 132:1 (1972)].In an attempt to determine the physiologic significance of this peptide, we rapidly removed the pituitaries from intact and adrenalectomized rats which were stressed by ether (2 minutes) or a buzzer (30 minutes). The neurointermediate lobes were separated from the anterior lobes and prepared for electron microscopy and immunocytochemistry (with the unlabeled antibody peroxidase-antiperoxidase complex technique) and ACTH bioassay (with the use of dispersed adrenal cells according to Sayers et al.) and radioimmunoassay.

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