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fixing cells/ Try the BioSci archives

'Mike' Michael J. Moser moser at U.WASHINGTON.EDU
Thu Mar 12 01:59:52 EST 1998


Chris

Before I give my answer I would just like to plug the BioSci archives. 
You can find out a lot about fixing GFP and many other subjects of
interest by pointing your web browser to: 

http://www.bio.net/
OR
http://www.bio.net/hypermail/FLUORESCENT-PROTEINS/

I fix fibroblasts that express S65T-GFP in chamber slides by replacing the
culture medium with 4 % formaldehyde with or without 0.1 -0.2 %
glutaraldehyde in 1 x PBS for 5 min at RT. 

Replace with 1 x PBS, 0.1% Triton for 2 minutes.

Proceed with immunofluorescent detection of myc epitope tagged antigen
using Texas Red secondary. 

Stain with 1 ug/ml DAPI and mount in aqueous mounting medium.

Longer fixation can still work, but GFP fluoreescence is decreases with
increased fixation.  In the archive also I found some reports that solvent
fixation has worked for some people.  In general I have found aldehyde
fixation to be the method of choice if your antigen permits it. 

I also find you can get away with using nail polish to seal if you make
sure there is sufficient mounting media to keep polish from contacting the
sample.  (If capillary action draws any polish under the coverslip, sample
is toast!)  Fancier methods are probably superior.

Happy GFPing

Mike

Mike Moser                                            Tel: 206-543-6585
UW Department of Pathology                            FAX: 206-543-3967
Box 357705                                       moser at u.washington.edu
Seattle, WA  98195                 http://weber.u.washington.edu/~moser

On 11 Mar 1998, Chris Lau wrote:

> 
> Hi,
> 
> I know this question has been asked a thousand times, but here is it
> again.
> 
> What fixative procedure can preserve the EGFP fluorescence and in the
> meantime suitable for immunohistochemistry with another antibody?  I don't
> mind taking pictures of the EGFP fluorescence first before the
> immunohistochemistry procedure.  I just need to show EGFP and
> immunohistochemistry on the same cells (on culture dishes)..
> 
> Thanks in advance.
> 
> 
> Chris Lau
> 
> _____________________________________________________________________________
> 
> Chris Lau, Ph.D. 				Tel: 415 476-8839 
> Division of Cell and Developmental Genetics 	FAX: 415 502-1613 
> Department of Medicine, VAMC-111C5 
> University of California, San Francisco 	E-mail : clau at itsa.ucsf.edu 
> 4150 Clement Street
> San Francisco, CA 94121
> 
> 
> 
> 
> 




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