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Low-fading immunofluorescence with propidium iodide contrast compared with immunogold light microscopy in whole cells and semi-thin cryosections

H Jensen, N Broholm and B Norrild

Institute of Medical Microbiology and Immunology, University of Copenhagen, Panum Institute, Denmark.

Conventional immunofluorescence produces excellent labeling but has drawbacks such as fading and the need for phase-contrast. Silver- enhanced colloidal gold probes allow counterstaining and permit permanent preparations with no fading if mounted correctly, but the most common limits of this technique are steric hindrance and the artifacts produced by silver enhancement. Our goal was to investigate Herpes simplex virus type 1 (HSV-1) morphogenesis by immunogold cryosection electron microscopy. We therefore needed a sensitive and reproducible immunocytochemical light microscopic method to confirm the immunofluorescence results in whole cells and to screen the cryopreparations before the time-consuming electron microscopic studies. We report data showing that the use of p-phenylenediamine to retard fading and propidium iodide to provide counter-staining results in brilliant fluorescence and contrast, minimal autofluorescence, and invisible fading at least for 5 min exposures, even in preparations with weak antigen presentation. Storage at -20 degrees C provides stable fluorescence. This method is superior to silver-enhanced colloidal gold light microscopy in our investigations.

Volume 43, Issue 5, pp. 507-513, 05/01/1995
Copyright © 1995 by The Histochemical Society


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H. L. Jensen and B. Norrild
Herpes simplex Virus Type 1-infected Human Embryonic Lung Cells Studied by Optimized Immunogold Cryosection Electron Microscopy
J. Histochem. Cytochem., April 1, 1998; 46(4): 487 - 496.
[Abstract] [Full Text]



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