How to fix injected embryos ?
halfonm at MINERVA.CIS.YALE.EDU
Fri Oct 13 11:51:10 EST 1995
A protocol for doing this is described by Prokop and Technau in the book
"Cellular Interaction in Development: A Practical Approach." Essentially,
prepare fix from a 1:1 mix of n-heptane: 25% glutaraldehyde. Shake well
and allow the phases to separtate. You can then apply just the heptane
phase to your embryo on the cover slip or in a small dish. If your
embryos are under oil, it helps if you remove as much if the oil as you
can. I find that a 10-15 minute fixation is sufficient.
The embryos must then be manually devitellinized. I find this difficult
to do using the Prokop and Technau protocol because the embryos tend to
come off of the adhesive during fixation. What I usually do is swith to a
technique described by Vincent and O'Farrell (Cell 68:923-931). The
embryo is transferred with a cut-off 200ul pipet tip onto the outside of
a fine mesh (e.g., nitex) basket stuffed with kim wipes. To prevent the
embryo from sticking to the tip, rinse the tip a few times in PBS with a
little serum in it first. Then use a piece of double-stick tape to
transfer the embryo to a small dish, cover with PBS, and devitellinize
with a sharp needle or forceps (this may take a little practice). You can
then proceed with antibody or lacZ staining or whatever.
I find this the easiest way to do things when I'm working with individual
embryos which I want to keep separate. If you're doing large numbers, you
may want to modify it a little. At any rate, some variation of the two
protocols I've mentioned ought to work for you.
(marc.halfon at yale.edu)
On Tue, 10 Oct 1995, Sidney B. Cambridge wrote:
> I am in the process of learning how to fix and antibody-stain injected
> embryos. All of the protocols I found call for fixation of the embryos
> while being attached to the coverslip. Besides wondering if there isn't
> any other way of fixing, I can't figure out how exactly the coverslip is
> placed in a biphasic solution
> (PEM/heptane) ????? Can anybody help me ?
> Thanx in advance.
> Sidney (sc6c at andrew.cmu.edu)
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