[Histonet] Autofluorescence

Mikael Niku 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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