GFP+fixation AND Ascaris homologue searches
Morris_maduro at biology.ualberta.ca
Mon Nov 20 15:21:38 EST 1995
GFP + FIXATION
I have tried methanol/acetone fixation followed by primary binding of a
monoclonal anti-Bgal, and secondary with a Texas-Red conjugated anti-mouse.
The nematodes were transgenic for a rescuing fusion of unc-119::GFP, and a
non-rescuing fusion of unc-119::lacZ.
It worked very well. The GFP fluorescence looks great after this kind of
fixation. (In fact, the worm whose image appeared in the Science worm
poster had been fixed with acetone before photography.) The problem is,
although the antibody signal can remain stable over the long term, the GFP
fluorescence slowly disappeared over a few days - so I had to take pictures
quickly. I have previously posted an inquiry about antibodies to GFP for
this purpose (which seems redundant) but didn't get many useful replies
(apparently some companies do make anti-GFP). However, the newer GFP may
give a more stable signal, and may be superior since the new GFP does not
fluoresce as well under the DAPI filter (allowing more control of double
exposures, for example).
Has anyone successfully obtained an Ascaris homolog from a C. elegans or C.
briggsae probe (using low-stringency hybridization)? If so, what were the
conditions? I am unable to use degenerate-oligo PCR since I do not know
where conservation should be for unc-119.
For those who have found homologues of their favorite genes in Ascaris,
what was the percentage amino acid conservation?
Thanks in advance,
Dept of Biological Sciences
University of Alberta
Edmonton, AB Canada morris_maduro at biology.ualberta.ca
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