Hi Rebecca,
What filter sets are you using to look at GFP? According to the Chalfie
paper describing gfp use as a reporter, excitation with short wavelength
light causes rapid bleaching. This is consistent with what I see. I
usually use two different filters for GFP. My favorite filter set, because
it gives a brighter signal, for looking at worms uses 400-440 excitation,
455 dichroic and ~470 long pass emission filter. I only use this filter
set with low magnification because when using it at high magnification I
get very rapid photobleaching. With a 40X dry objective the signal
bleaches in 20 to 30 seconds. When I change to a 100X oil objective the
signal bleaches in ~5 seconds. I switch to a FITC filter set (450-490
excitation, 510 dichroic and 520 long pass) for most photography. I am not
sure crossing into an Unc strain would help because the animals would
probably still move a little while you were exposing the film (I'm usually
using ~30 second exposures, although this can be reduced by using a faster
film). If they did move, obviously your pictures would be blurred. Also, I
see photobleaching even in the absence of azide, although it is less
rapid. I would just try a different filter set, which hopefully will take
care of your bleaching problem.
Your second question is much more difficult to answer. I think in
practice, if one sees robust expression in a pattern that makes sense for
the gene in question, you tend to believe it. It's generally preferable to
confirm the expression pattern with antibody staining once that becomes
available. I think most reporter experiments are taken with a grain of
salt. Obviously, your mosaic analysis will help interpret the expression
pattern you see.
Hope this helps.
Wayne Forrester
