You might want to try Sudan Black as an alternative to borohydride and
CuSO4. You can find an appropriate and reproducible suppression of
autofluor by testing a dilution series starting with 0.1% in 70% ethanol
(Journal of Histochemistry and Cytochemistry, Vol. 49, 1565-1572,
Control of Autofluorescence of Archival Formaldehyde-fixed,
Paraffin-embedded Tissue in Confocal Laser Scanning Microscopy
(CLSM),Werner Baschong, Rosmarie Suetterlin, and R. Hubert Laeng). I've
had pretty good luck with this on free-floating rat brain sections and
GFP, usually with dilutions of the 0.1% by 10-100X. If you start with
100X diluted 0.1% and it isn't enough you can put the section back in a
higher concentration until its right. Kind of a nice counterstain for
showing tissue structure (fiber tracts) too.
For postfix we refrigerate the whole perfused (100 ml wash, 400 ml fix)
rat 2 hr. before removing the brain into 30% sucrose PBS. It gives the
fixative a bit more time to work without impairing immunolabeling for
most antibodies. The glycerol/glycol may be responsible for some of the
gooeyness of your sections, too.
UF Pharmacology & Therepeutics
Date: Tue, 29 Apr 2008 09:54:19 -0400
From: "Spitzer, Nadja"
Subject: [Histonet] rat brain autofluorescence
...extreme autofluorescence of the sections...the treatments are pretty
hard on my free floating sections...
Thanks very much I appreciate your input.
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