Optimal visualization of immunogold-silver staining of hepatic PEPCK with epipolarized light microscopy

We used immunogold-silver staining to localize phosphoenolpyruvate carboxykinase in 10 microns cryosections of 4% paraformaldehyde perfusion-fixed normal male rat liver. The resolution and sensitivity of detection were improved by epipolarized light microscopy of 0.5 microns semi-thin plastic sectio...

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Bibliographic Details
Published in:The journal of histochemistry and cytochemistry 1994-06, Vol.42 (6), p.823-826
Main Authors: Gao, K, Giffin, BF, Morris, RE, Cardell, EL, Cardell, RR
Format: Article
Language:English
Subjects:
Animals
Antibodies
Biological and medical sciences
Diverse techniques
Fundamental and applied biological sciences. Psychology
Gold
Histological Techniques
Immunohistochemistry - methods
Liver - cytology
Liver - enzymology
Male
Molecular and cellular biology
Phosphoenolpyruvate Carboxykinase (GTP) - analysis
Rats
Rats, Sprague-Dawley
Silver
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Summary:We used immunogold-silver staining to localize phosphoenolpyruvate carboxykinase in 10 microns cryosections of 4% paraformaldehyde perfusion-fixed normal male rat liver. The resolution and sensitivity of detection were improved by epipolarized light microscopy of 0.5 microns semi-thin plastic sections prepared from these pre-embedding immunogold-silver-enhanced 10-microns thick cryosections. Epipolarized light combined with transmitted light simultaneously demonstrated antigenic sites (visualized with epipolarized light illumination) and tissue morphology (revealed by transmitted light). To optimize the conditions for high resolution, an oil immersion objective lens (x 100) with adjustable iris diaphragm was used with different intensity settings for both light sources. Our observations indicate that if the intensity of the transmitted light is too high, the visibility of the gold-cored silver grains by epipolarized illumination is decreased; if the intensity of epipolarized light is too strong, haloes appear around the gold-cored silver particles. By adjusting the aperture in the objective lens and the neutral density filter in the transmitted light pathway to balance the intensities of transmitted and epipolarized light, an optimal image is obtained that shows the maximal number of antigenic sites and excellent morphology.
ISSN:0022-1554
1551-5044
DOI:10.1177/42.6.8189043