OPTIMAL VISUALIZATION OF IMMUNOGOLD-SILVER STAINING OF HEPATIC PEPCK WITH EPIPOLARIZED LIGHT-MICROSCOPY

We used immunogold-silver staining to localize phosphoenolpyruvate car boxykinase in 10 mu m cryosections of 4% paraformaldehyde perfusion-fi xed normal male rat liver. The resolution and sensitivity of detection were improved by epipolarized light microscopy of 0.5 mu m semi-thin plastic sections prepared from these pre-embedding immunogold-silver-e nhanced 10-mu m thick cryosections. Epipolarized light combined with t ransmitted light simultaneously demonstrated antigenic sites (visualiz ed with epipolarized light illumination) and tissue morphology (reveal ed by transmitted light). To optimize the conditions for high resoluti on, an oil immersion objective lens (x100) with adjustable iris diaphr agm was used with different intensity settings for both light sources. Our observations indicate that if the intensity of the transmitted li ght is too high, the visibility of the gold-cored silver grains by epi polarized illumination is decreased; if the intensity of epipolarized light is too strong, haloes appear around the gold-cored silver partic les. By adjusting the aperture in the objective lens and the neutral d ensity filter in the transmitted light pathway to balance the intensit ies of transmitted and epipolarized light, an optimal image is obtaine d that shows the maximal number of antigenic sites and excellent morph ology.