A double-immunostaining procedure using colloidal gold, ferritin, and peroxidase as markers for simultaneous detection of two hypophysial hormones with the electron microscope.

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Abstract
For simultaneous ultrastructural localization of intracellular peptides, protein A-gold techniques or immuno-gold techniques have generally been applied. The present study reports a double immunostaining procedure for simultaneous visualization of two hypophysial hormones (prolactin and corticotropin) on a single ultrathin section of the pars distalis of an amphibian. Prolactin and corticotropin antisera were respectively raised in guinea pigs and rabbits and were applied simultaneously to ultrathin sections. Antigenic binding sites were detected under the electron microscope using differently labeled species-specific secondary antisera raised in goats or sheep. Three labels (gold particles, ferritin, peroxidase) were checked for double labeling. The combinations investigated were: 1) two gold preparations or IgG-gold labeled with different-sized gold particles; 2)IgG-gold and IgG-ferritin; 3) IgG-gold and IgG-PAP (peroxidase-antiperoxidase). The double-immunostaining procedures described here have proved useful in the simultaneous ultrastructural localizations of two intracellular antigens on a single tissue section. These procedures constitute a basis for the development of triple immunostaining methods.