scholarly journals IDENTIFICATION OF ACTH CELLS AND TSH CELLS IN RAT ANTERIOR PITUITARY WITH THE UNLABELED ANTIBODY ENZYME METHOD ON ADJACENT THIN AND THICK SECTIONS

1980 ◽  
Vol 13 (6) ◽  
pp. 627-645 ◽  
Author(s):  
YASUYUKI KAWARAI
Keyword(s):  
Anterior Pituitary ◽  
Tsh Cells ◽  
Enzyme Method ◽  
Unlabeled Antibody

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 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.

scholarly journals A COMPARISON OF ALCIAN BLUE-ALDEHYDE FUCHSIN AND PEROXIDASE-LABELED ANTIBODY STAINING TECHNIQUES IN THE RAT ADENOHYPOPHYSIS

1970 ◽  
Vol 18 (6) ◽  
pp. 450-454 ◽  
Author(s):  
ALICE E. SWOPE ◽  
RAYMOND H. KAHN ◽  
JAMES L. CONKLIN
Keyword(s):  
Alcian Blue ◽  
Anterior Pituitary ◽  
Anterior Lobe ◽  
Female Rats ◽  
Intermediate Lobe ◽  
Antibody Staining ◽  
Aldehyde Fuchsin ◽  
Tsh Cells ◽  
Staining Techniques ◽  
Lh Cells

The peroxidase-labeled antibody (P-Ab) technique was compared on adjacent sections with a permanganate-Alcian Blue (AB)-aldehyde fuchsin (AF) procedure on the anterior pituitary gland of young adult, female rats. The cells that stained with both AB and AF (AB, AF+) were large and polygonal and frequently possessed long processes; these cells correspond to those which reacted with the TSH antibody. The cells that stained only with AF reacted with the FSH antibody in the P-Ab technique and the cells which reacted with the LH antibody were not stained with either AB or AF.AB,AF+ cells ("TSH" cells) were distributed throughout the anterior lobe except along the lateral and dorsal peripheries of the gland and adjacent to the intermediate lobe, while both the AF+ ("FSH" cells) and the "LH" cells were distributed throughout the anterior lobe.

scholarly journals Adrenal luteinizing hormone releasing hormone receptors.

1978 ◽  
Vol 26 (8) ◽  
pp. 613-617 ◽  
Author(s):  
L A Bernardo ◽  
J P Petrali ◽  
L P Weiss ◽  
L A Sternberger
Keyword(s):  
Luteinizing Hormone ◽  
Hormone Receptors ◽  
Solid Phase ◽  
Pyroglutamic Acid ◽  
Zona Fasciculata ◽  
Paraffin Sections ◽  
Luteinizing Hormone Releasing Hormone ◽  
Releasing Hormone ◽  
Enzyme Method ◽  
Unlabeled Antibody

Paraffin sections of mouse adrenals processed with antiserum to luteinizing hormone-releasing hormone (LHRH) in the unlabeled antibody enzyme method reveal moderate staining in the cytoplasm of cells of zona fasciculata and reticularis. The stain is intensified upon pretreatment of sections with LHRH. Pretreated sections processed with solid phase immunoabsorbed LHRH are unstained. Analogues of LHRH deficient in the C-terminal glycine amide inhibit staining, while analogues deficient in the N-terminal pyroglutamic acid have no effect. It is concluded that the adrenal contains receptors for a ligand resembling LHRH in receptor and immunoreactivity. The possibility is considered that the ligand may be an inhibitor of pineal origin.

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

1975 ◽  
Vol 23 (9) ◽  
pp. 666-677 ◽  
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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