Hi again --
I sent this question about GFP around before the holidays. I thought
other people might find the many responses useful, so I have
summarized them here. I've included a couple emails in their entirety
(with permission from their authors) as they had a lot of detailed
info. Thank you all so much for replying so quickly!!
Liz Ryder
(my original question follows)
Hi --
I am trying to figure out what cells my gene of interest is
expressed in, using a GFP fusion. In order to help figure out what
cells I'm seeing expression in, I want to do a double-labeling
experiment with my GFP fusion and an antibody stain (to LIN-26) as a
marker. I'm assuming GFP is killed by fixation; am I right? And if
so, does anyone have advice about what commercially available
anti-GFP antibodies work well to stain embryos?
Thanks for your help!
Liz Ryder
The general consensus was that GFP is not typically completely killed
by fixation, but the intensity may be reduced. Sometimes the
reduction is not a problem, but other times, it renders the GFP
staining useless. Seems likely that this depends on how bright the
staining was to begin with. Keep animals out of the light.
Antibodies from a number of companies work (see below).
=46ixatives used successfully (retaining some or most GFP staining):
=46inney-Ruvkun protocol
methanol/acetone
1-4% paraformaldehyde
50% MeOH
5 minutes in RT MeOH (for embryos)
Anti-GFP antibodies used successfully:
Chemicon: Chk x GFP AB16901 (used 1:200) The best one, according to
Morris Maduro!
(for secondary, Morris uses Jackson ImmunoResearch:
Donkey Anti-Chicken IgY 703-095-155 (used 1:200)
details below
Clontech (several labs used, mostly successfully)
one lab mentioned rabbit polyclonal worked well
two labs mentioned monoclonal worked well (Clontech catalog
number 832-1)
one lab did not specify which antibody they used
one lab mentioned that the polyclonal peptide Ab did not work
well for them)
RDI (for immunoEM, not with GFP)
Molecular Probes mouse anti-GFP 3E6 (1:100)
More detailed replies:
A student of mine, Jose Fos, has done some really nice double
stainings with anti-GFP and anti-CEH-13 antibodies. He did stain
freeze-cracked acetone fixed embryos with a monoclonal anti-GFP
antibody (Clontech catalog number 832-1) and an FITC conjugated
secondary antibody (Jackson Immunoresearch Lab. catalog number
315-095-003). In our hands GFP is severely weakened but not
completely killed by fixation. It is too weak to still be useful but
strong enough to bother you if there is something else that is
labelled in green. We therefore use a green (FITC conjugated)
secondary to detect GFP and a red (Cy3) conjugated one to detect
CEH-13. This way, for GFP, the fluorescence and the signal from the
antibody staining enhance each other and the remaining green
fluorescence does not interfere with the signal from CEH-13 that
appears in red.
(Adrian can be contacted for the protocol).
Adrian Streit, Ph.D.
Department of Biology/Zoology
University of Fribourg Perolles
Ch. du mus=E9e 10
1700 Fribourg, Switzerland
Phone: ++41 26 300 88 65
=46ax: ++41 26 300 97 41
e-mail: Adrian.Streit at unifr.ch
Hi Liz,
Not all fixation procedures kill GFP fluorescence entirely. I routinely use
either the Finney-Ruvkun procedure (available on the Ambros protocol page)
or methanol/acetone, and the chicken anti-GFP polyclonal from Chemicon, to
stain embryos. I've tried many other anti-GFP Abs and this is the best one.
I use an FITC-conjugated anti-chicken secondary from Jackson labs. The FITC
filter is used for the anti-GFP because any remnant GFP fluorescence makes
the channel useless for anything else. Both the primary and secondary are
used at ~1:200, and depending on the fusion, staining can be done at room
temperature for an hour.
Chemicon: Chk x GFP AB16901
Jackson ImmunoResearch: Donkey Anti-Chicken IgY 703-095-155
Alternatively, you could generate multi-color GFP transgenes. I have a
lin-26::CFP; you could make a YFP fusion with your other gene, and bring two
strains together. Of course, you need the special filter sets to
discriminate the two, etc.
Good luck,
Morris
Morris Maduro (Rothman Lab)
Dept. MCD Biology
UC Santa Barbara
Santa Barbara, CA 93106
(805) 893-8090
fax (805) 893-2005
Liz Ryder
Dept. Biology and Biotechnology
Worcester Polytechnic Institute
100 Institute Rd.
Worcester, MA 01609
TEL: 508-831-6011
=46AX: 508-831-5936
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