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visualising EGFP in vivo

megason at my-deja.com megason at my-deja.com
Wed Jul 14 11:50:11 EST 1999


I've had experience using an IRES-EGFP and
straight EGFP construct in mice and chicks. Both
can be detected in whole mount or cryosections. I
routinely fix overnight in 4% paraformaldehyde /
PBS+0.1% Triton before cryosectioning. Fixing
doesn't affect brightness and you get much better
histology. Once fixed, the slides can be treated
with methanol if needed and still fluoresce. With
the strong promoter I'm using, the IRES is at
least half as bright as the non-IRES construct.

Also GFP is extremely stable, so I wouldn't
stress too much about delay before viewing. I
have embryos stored in 80% glycerol for months
that are very bright. The GFP in my tail preps is
even still fluorescent after the tails are turned
to mush by ProK and 55'C overnight.

As far as the autofluorescence in neurons you're
seeing, I think you'll know the GFP expression
when you see it. It is a very defined shade of
green. Autofluorescence seems to have a
broader/yellowish spectrum.

Also what PCR primers are you using for
genotyping? I've tried developing some but they
gave false positives.

Sean Megason


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