[Histonet] rat brain autofluorescence


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.
Good luck,
Mike King
UF Pharmacology & Therepeutics

Date: Tue, 29 Apr 2008 09:54:19 -0400
From: "Spitzer, Nadja" 
Subject: [Histonet] rat brain autofluorescence

Hello All,
...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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