There's several things I don't understand:

(1)  Why don't you use a CY3 secondary?  Why are you going through this 3-step
procedure, thereby increasing any backgound problems you might have?

(2)  Have you done all the requisite controls to investigate whether this is
truly autofluorescence and not a background problem?  In otherwords, is the
tissue still fluorescent when you eliminate your fluorescent secondary?

(3)  If the answer to (2) is yes, you might try a different mounting medium.
In fish, autofluorescence dramatically increases if you mount in nonaqueous
medium, presumably because water quenches the endogenous fluorescence of the
tissue.  Therefore, if you dehydrate the tissue, you get this screaming
autofluorescence.  You really do have to stick with an aqueous-based mounting
to attenuate the autofluorescence in fish.  Computer-based archiving is
required.  Maybe the same is true with your tissue.

(4) If the answer to (2) is no, you need to do all possible negative controls,
eliminating the primary is one set of experiments, eliminating the
secondary in
another, etc.  Once you identify the source of the background, you then
need to
carefully titer down the offending reagent until background staining is
eliminated.  If the offending reagent is your primary, and it's a polyclonal,
you may not be able to eliminate the background.  You may want to try an
affinity-purified primary instead.  Sometimes, however, affinity-type
purifications won't eliminate the background problems with a polyclonal.  If
available, switch to a monoclonal.  If your offending reagent, is your
fluorescent antibody, the reagent may be bad.  It may be partially denatured
for some reason, thereby readily sticking nonspecifically to your tissue.

I hope this helps.

Karen Larison




Date:          Tue, 22 Dec 1998 08:54:56 -0500
From:          "Michael J. Lyon, Ph.D." <lyonm@VAX.CS.HSCSYR.EDU>
Subject:       Autofluorescence
To:            "Histonet (E-mail)" <HistoNet@Pathology.swmed.edu>

Hello:

I have just started doing immunostaining of muscle dissections.  The tissue
has
been immersion fixed in 4% paraformaldehyde 0.2% Picric acid for about 24 hrs
at 4C.  My primary antibodies are all fa
rly dilute at 1:3000 or 1:4000, secondary is at 1:400 and I am using CY3 at
1:600.  I have tried ammonium chloride and sodium borohydride but still have
considerable background from the muscle fiber
.  I was thinking of switching to a different fixation.  Any suggestions would
be appreciated.  Hope I have given sufficient info.

Thanks in advance

Michael