RE: GFP (visualization)
The answer to your question is "It depends". If the investigator who is
introducing the GFP reporter uses the EGFP (enhanced) version or one of its
variants you can paraffin process and still see the fluorescence. If you are
comfortable cutting frozen brain, by all means utilize the native
fluorescence. If you feel fixation is necessary use an aqueous fixative like
formalin. That way you don't have to worry about the alcohol stability of
Green fluorescent protein as originally used did not withstand alcohol
dehydration and required either post-processing visualization by IHC with
anti-GFP or more recently by In situ PCR amplification.
We have injected viral vectors containing EGFP (from Clontech) into fracture
sites in our mouse models for fracture healing. Then harvested the bone,
formalin fixed them, decaled in 5 % formic acid and paraffin processed. Cut
sections on standard slides deparaffinized in xylene 3 x 5 min each
coverslipped and taken the slide to the fluorescent scope for visualization.
The advent of EGFP variants of differing wavelengths (yellow, red and now
blue)are allowing our investigators to use two viral vectors and visualize
the location of the fluorescence in two colors.
Refer to Clontech's catalog for extensive information on GFP stability and
that of its variants.
Good luck, Donna Montague
UAMS Medical Center
Little Rock, AR
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