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Protocol: Rehydrating embryos once fixed and dehydrated…

Posted by , on 9 July 2010

Does anyone know a protocol for rehydrating embryos once stored in methanol?  I’m working with Xenopus laevis embryos which have been fixed using formaldehyde.  Some lab wisdom passed down is “wash into a 50:50 solution of 1xPBS and methanol; then into 1xPBS; then into methanol again (which we’re a bit confused by); then into 50:50 PBS: methanol; and finally 1xPBS”.

Any help would be much appreciated!

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3 thoughts on “Protocol: Rehydrating embryos once fixed and dehydrated…”

  1. Hi Gary.
    We used to fix mouse embryos (both pre- and post-implantation) in PFA then methanol. After your standard PFA fix, wash in PBS then pass them through 25%, 50%, 75% then 100% methanol (in PBS, 5mins each step). To rehydrate, just do the reverse: 75%, 50%, 25% then back into PBS. This worked fine for me and others in the lab (the embryos do get a little sticky while in the methanol, though!).
    Hope this helps

  2. Thanks Seema!
    I’ve just carried it out as you suggested – my main problem was that I coinject embryos with beta-galactosidase and then stain to check for injection using X-gal, but had forgotten to do this before dehydrating embryos.
    The staining is taking much longer but does seem to be working – thank you!

  3. Gary – what Seema suggested is exactly what we do with Xenoups laevis embyros! So it should work fine! (ps only fix in formaldehyde ((memfa)) for one hour when doing x-gal staining) Ruth

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