Liisa,

There are a couple of simple things you can do to help reduce autofluorescence.
Some of the chemical reactions causing autofluorescence occur most rapidly with
higher temperatures and on exposure to light. Therefore, performing the labeling
at 4 C in the dark can help reduce this problem.

Autofluorescence intensity is related to section thickness. You may want to try
thinner sections if at all possible.

Sometimes using fluorophores excited at longer wavelengths can help diminish
autofluorescence.

If autofluorescence is still an issue, there are a few preincubation steps you
could try.

A Tris-glycine mixture (adjust 0.1M glycine to pH 7.2-7.4 with 1M Tris base)
will saturate free aldehyde groups. (15-30 minutes at room temp in Tris-glycine.
Wash well in PBS)

The use of 1% sodium borohydride in PBS helps reduce any free aldehyde groups in
the tissue, making them nonreactive. Incubate sections for 30 minutes in
borohydride and then wash well(minimum 15 minutes) in several changes of PBS.
Proceed with labeling.

These techniques can be used alone or sequentially. If the tissue is fragile
though, only use the Tris-glycine method.

Please note that sodium borohydride is very reactive and is flammable on contact
with water

Another technique to block unreacted groups is to incubate sections for 5
minutes in 50mM NH4Cl, and rinse in PBS before labeling.

Hope this helps

Ronnie Houston
Regional Histology Operations Manager
Bon Secours Health Partners Laboratories
5801 Bremo Road
Richmond, VA 23226


______________________________ Reply Separator
_________________________________
Subject: Re: abolishing autoflorescence
Author:  Liisa Tremere  at BSHSIBTW
Date:    6/20/02 1:41 PM




Dear Histonetters,

Does anyone know of a protocol that would allow us to reduce the autoflorescence
in CNS tissue caused by perfusing with gluteraldehyde?
 Any advice would be greatly appreciated.

Thanks in advance,

Liisa