Kx. Gao et al., OPTIMAL VISUALIZATION OF IMMUNOGOLD-SILVER STAINING OF HEPATIC PEPCK WITH EPIPOLARIZED LIGHT-MICROSCOPY, The Journal of histochemistry and cytochemistry, 42(6), 1994, pp. 823-826
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.