[Histonet] Help me get rid of autofluorescence

[Gayle Callis]

Unfortunately, you have to live with it.  Anytime you fix tissue with 
paraformaldehyde or NBF, you will increase autofluorescence.  Sometimes it 
is better to snap freeze fresh tissue,cryosection and fix with formalin 
vapors. Obviously, this is not possible for you, OR you can detect the GFP 
with immunohistochemistry or immunofluorescence using a different colored 
fluorophore.  This was discussed this past summer at great length with one 
gentleman from Germany who succeeded in using the formalin vapor method. I 
think I also have that as a pdf and will send to you.  Not sure about 
DsRed, but maybe a red GFP chimera is a better choice and use the 
autofluorescence like a counterstain???

I have a review of autofluorescence in conjunction with GFP  I am going to 
attach to you privately.  A very interesting paper.

At 03:06 PM 9/29/2004, you wrote:
>I have a virus that expresses GFP during replication.  I have fixed tissue
>from animals
>infected with the virus in 4% paraformaldehyde (Paraformaldehyde from Ted
>Pella as a
>16% solution) in water.  I fix tissues for 4 hours, soak 1-2 days in 30%
>sucrose, freeze in
>OCT, and then section on the cryostat.  My control uninfected tissue samples
>have high
>fluorescence.  Is there a way to avoid this autofluorescence?  Keep in mind
>that I have to
>fix the samples to kill the virus (dangerous to man).
>Thanks in advance,
>Justin
>
>Institute for Antiviral Research
>Biotechnology Center Rm. 201
>4700 Old Main Hill
>Utah State University
>Logan, UT 84322-4700
>Fax (435)797-3959
>Ph. (435)797-3643
>jgjulander <@t> cc.usu.edu
>
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)