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

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 hor...

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Bibliographic Details
Published in:The journal of histochemistry and cytochemistry 1975-09, Vol.23 (9), p.666-677
Main Authors: Erlandsen, SL, Parsons, JA, Burke, JP, Redick, JA, Van Orden, DE, Van Orden, LS
Format: Article
Language:English
Subjects:
Animals
Cross Reactions
Female
Glucagon - analysis
Glucagon - immunology
Growth Hormone - analysis
Growth Hormone - immunology
Immunoelectrophoresis
Insulin - analysis
Insulin - immunology
Male
Mammary Neoplasms, Experimental - analysis
Microscopy, Electron
Pancreas - analysis
Pancreas - ultrastructure
Pituitary Gland - analysis
Pituitary Gland - ultrastructure
Radioimmunoassay
Rats
Subcellular Fractions - analysis
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Summary: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.
ISSN:0022-1554
1551-5044
DOI:10.1177/23.9.1176760