Re: [Histonet] GFP
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.
>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
>Department of Physiology - Room 541
>Medical College of Wisconsin
>8701 Watertown Plank Road
>Milwaukee, Wisconsin 53226
>Histonet mailing list
Veterinary Medicine - Anatomy, Physiology and Cell Biology
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Davis, CA 95616
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