mikael.niku <@t> helsinki.fi
Mon Nov 13 01:21:10 CST 2006
I tried the photobleaching method after reading the paper about it, but
couldn't make it work. Briefly, I purchased fluorescent tubes with
specific wavelengths and exposed paraffin sections to them up to 2-3
days or so, with absolutely no observable effect at all. Tried several
wavelengths, no success. Perhaps I was doing something wrong, but I
suspect the energy should be much higher (I think the authors actually
recommended using an epifluorescence microscope with a low-power
objective for best results; for large numbers of slides, this is
obviously not feasible).
I have got great results with 0.1% Sudan Black B (CI 26150) in 70%
ethanol (for PFA-fixed, paraffin-embedded 4 um sections of various
bovine tissues), applied for 15-30 min after the fluorescent secondary
antibody. Note that the stuff takes time to dissolve.
With best regards,
Stephen Clark wrote:
> My name is Stephen Clark and i work in the neuro lab at Eastern Illinois University. We use fluorescent immunohistochemistry to stain the Olfactory Epithelium of mice and have recently had a problem with autofluorescence of our tissues. They are fixed in 4% paraformaldehyde in PBS and i have already tried sodium borohydrate with little success. I have read about photobleaching using UV, neon, and lights of specific wavelengths (488 and 633nm). I am just wondering if anyone utilizes photobleaching via a light source and where it would be possible to purchase them. I am also wondering if the 18um sections might be a little too thick and whether that would have an increased affect on autofluorescence. Any tips would be appreciated.
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Mikael Niku URL: www.helsinki.fi/~mniku/
University of Helsinki Dept. Basic Veterinary Sciences
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