How to fix injected embryos ?

Marc Halfon halfonm at MINERVA.CIS.YALE.EDU
Fri Oct 13 11:51:10 EST 1995


Sidney:

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.

Good Luck!

Marc Halfon 
(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.
> 
> Cheers,
> Sidney   (sc6c at andrew.cmu.edu)
> 
> 



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