Re: Advice.

From:Karen Larison <>

<!doctype html public "-//W3C//DTD W3 HTML//EN"> <html><head><style type="text/css"><!-- blockquote, dl, ul, ol, li { margin-top: 0 ; margin-bottom: 0 } --></style><title>Re: Advice.</title></head><body> <div>Ian,</div> <div><br></div> <div>Good luck on this one!  TCA is an excellent fixative for whole mounts if it's compatible with your antibody.  TCA is known to cleave proteins.  My assumption is that it makes the whole mount more permeable, and this is why it works so well.  However, after TCA fixation, the embryo is much, much more fragile.  Zebrafish embryos tend to shred like toilet paper after TCA fixation, and you have to handle them very, very gently.  The fixation time should be kept short (or the tissue will literally fall apart), and ideally the IHC process begun immediately!  Have your researchers tried Dent's fixative?  I have found that if an antibody works with TCA, it will also usually work with Dent's.  The embryos will be far less fragile if you fix in Dent's, and then stored in ice-cold methanol.  Dent's fixative is described in "Whole-Mount Staining of Xenopus and Other Vertebrates", MW Klymkowsky, Methods in Cell Biology, 36, 419-441 (1991).  It's 20% DMSO/80% methanol.  HANDLE WITH CARE!!</div> <div><br></div> <div>Karen in Oregon</div> <div> </div> <div><br></div> <div><br></div> <div><br></div> <blockquote type="cite" cite><x-tab>        </x-tab>I have a client who is going to Trinidad for 4 weeks collecting Xenopus larvae then bringing them back to Glasgow for whole mount staining. We will be looking for Tyrosine Hydroxylase in hatching glands after fixation in 2% trichloroacetic acid/PBS. 2% TCA is the fix of choice, others have been tried but in this instance TCA is the preferred method.<br> What I'm not sure about is handling after fixation. Do I leave in TCA for the 4 weeks or place in PBS until they come to Glasgow. This is a field trip so further processing is not possible, it has to be fix or fix then buffer before the flight back to Scotland.<br> <x-tab>    </x-tab>All this because my microscopy brief was expanded. Wish it was the wallet and not just the waist line that is expanding.<br> Ian.   <br> </blockquote> <blockquote type="cite" cite><font color="#0000FF">Dr. Ian Montgomery,<br> West Medical Building,<br> University of Glasgow,<br> Glasgow,<br> G12 8QQ.<br> Tel: 0141 339 8855.  Extn:6602.<br> Fax: 0141 330 2923<br> e-mail:</font></blockquote> <div><br></div> </body> </html>
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