Re: [Histonet] GFP

From:john coppens

i have been working with some mice that express GFP in the Clara 
Cells of the lung.  the signal is quite strong in the cells that are 
positive.  i have had positive results under the following situations:

Frozen Tissue, my experience was similar to yours.

Whole Tissue, 4% 2hr paraformaldehyde fixed  and unfixed.  these were 
viewed under PBS with immersion objectives.  The signal lasts for 
weeks when the tissue is fixed. I can look at it all day with no 
noticeable photo-bleaching. I only use the unfixed tissue to verify a 
that  mouse is positive for the protein.

Paraffin Embedded, fixed 2 hours in 4% paraformaldehyde, then 
transferred to 50% EtOH.  I would lower the temperature of the 
paraffin pot to 56% if you normally run it higher.  The signal is 
fair but bleaches easily.  If you want it stronger and longer lasting 
signal you can do IHC with a GFP antibody and fluorescent secondary.

GMA Embedded, I believe we followed our standard protocol for GMA. 
There was a signal but if I remember right, it bleached out very 
quickly in the 1um sections.

Good luck.

>Has anyone worked with GFP tissue?
>I have found GFP is surviving frozen sections, acetone and 100% alcohol.
>I am wondering about FFPE tissue.  I'm thinking the heat during processing
>would destroy the GFP.
>Does formalin fixation effect the GFP?
>Is it possible to fix the tissue in an alcoholic fixative and manually hand
>process at room temperature and infiltrate into plastic?   Would the plastic
>medium destroy the GFP?
>What about celloidin embedded tissue?  This is not my choice
>Any information would be helpful.
>I thank everyone on the histonet that has in the past and future helped me.
>I really appreciate your help.
>Carol Ann Bobrowitz
>Histology Laboratory
>Department of Physiology - Room 541
>Medical College of Wisconsin
>8701 Watertown Plank Road
>Milwaukee, Wisconsin   53226
>FAX 414-456-6546
>Histonet mailing list

John Coppens
Veterinary Medicine - Anatomy, Physiology and Cell Biology
1 Shields Ave.
UC Davis
Davis, CA 95616
Histonet mailing list

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