ENHANCEMENT OF ANTIGENIC SITE DETECTION WITH GOLD-LABELED SECONDARY AND TERTIARY ANTIBODIES USING THE IMMUNOGOLD-SILVER STAINING METHOD

We report a modification of the immunogold-silver staining method (IGS S) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primar y antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope exami nation of 10 mum frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling densit y (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy . The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shell s and less background. We achieved intense specific staining of hepato cytes expressing PEPCK while minimizing background staining. The use o f combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable met hod for use in both light and electron microscopy.