bolzer wrote:

> I have formaldehyd-fixed mouse cerebellum tissue sections to
> Fluorescence-In-Situ-hybridize.
> The sections are cut with a vibratom without parafin embedding,
> that means they are quite thick, about 20 um.
> In each of our fluorescence channels they exhibit an
> extraordinary autofluorescence, making it hard to see the signal.
> Is there a way of at least weaken the autofluorescence?
> I already tried CuSO4, but the autofluorescence didn't decrease.

There are several sources of autofluorescence.  Lipofuscin
autofluorescence is granular and intracellular; it increases with the
age of the animal.  (The trick with CuSO4 decreases lipofuscin
autofluorescence; the paper on that by Steve Schnell, Bill Staines and
me should be appearing in J Histochem Cytochem in or around the July
1999 issue).  However, what you're describing may be aldehyde-induced
autofluorescence.  Aldehyde-induced autofluorescence will generally be
more diffuse, more generalized, much less puntate, will increase with
the amount of time the tissue has been fixed, and may be greater with
glutaraldehyde fixation than with formaldehyde fixation.  Since it
appears to be due to the formation of Schiff bases (--nitrogen
double-bonded to carbon), people have used various reducing agents
(e.g., NaBH4) to break the double-bonds.  There've been several recent
papers using the technique:

 Clancy B.  Cauller LJ. Reduction of background autofluorescence in
brain sections following immersion in sodium borohydride. Journal of
Neuroscience Methods.  83(2):97-102, 1998

 Tagliaferro P.  Tandler CJ.  Ramos AJ.  Pecci Saavedra J.  Brusco A.
Immunofluorescence and glutaraldehyde fixation. A new procedure based on
the Schiff-quenching method. Journal of Neuroscience Methods.
77(2):191-7, 1997

 Beisker W.  Dolbeare F.  Gray JW. An improved immunocytochemical
procedure for high-sensitivity detection of incorporated
bromodeoxyuridine. Cytometry.  8(2):235-9, 1987

Good luck!

Martin Wessendorf

--
Martin Wessendorf, Ph.D., Associate Professor
Dept Cell Biology and Neuroanatomy              office: (612) 626 0145
University of Minnesota                         lab:    (612) 624 2991
Minneapolis, MN  55455                  Preferred FAX:  (612) 625 4498
e-mail: [log in to unmask]        Dept FAX:       (612) 624 8118