*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Doug--

On 6/4/2013 12:23 PM, Cromey, Douglas W - (dcromey) wrote:

> I am working with a lab looking at IF stained sections of formalin-fixed, paraffin embedded pieces of mouse or rat liver.  If there was any actual staining in their first try on our confocal, it was largely washed out by the lovely and strong autofluorescence.  Because their animal experiment takes 8 weeks, it's not trivial (or inexpensive) to get new tissue, so they have been using existing blocks.  No one in their lab had the foresight to put up snap-frozen tissues for cryosectioning.  I have them looking at this document: http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf but I'm wondering if liver is always a problem with autofluorescence, or is there maybe something in this lab's sample prep protocol that's making it worse.  Any ideas?

Several questions:

0)  Are you sure it's autofluorescence and not a problem with your
staining protocol?  Do you see the same autofluorescence in sections
that haven't been stained?
1)  What does the autofluorescence look like?  Is it small, bright dots
or a uniform high background?
2)  Can you see it with the fluorescein filter?  With the rhodamine
filter?  With UV excitation?
3)  What color is the autofluorescence under a wide-band UV filter?

The cure depends on the cause.  If you see a uniform high background,
it's probably due to fixation, and treating the sections with sodium
borohydride (NaBH4) as Jason Kilgore suggested should help.  See:
http://www.ncbi.nlm.nih.gov/pubmed/9765122

If the autofluorescence looks like small, bright dots that don't
photobleach, show up using all your filter sets and look orange using a
wide-band UV filter, it's probably lipofuscin.  That can be reduced by 
treating with Sudan Black, as Mark Sanders suggested, or with CuSO4, 
which is described in the same paper.  CuSO4 is the better choice if 
you're running your tissue through xylene after staining--the Sudan 
Black will be eluted by lipophilic solvents.

Good luck!

Martin Wessendorf
-- 
Martin Wessendorf, Ph.D.                   office: (612) 626-0145
Assoc Prof, Dept Neuroscience                 lab: (612) 624-2991
University of Minnesota             Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: (612) 626-5009
Minneapolis, MN  55455                    e-mail: [log in to unmask]