(no subject)

[Graham Thomas]

>Here's a simple question, which I hope someone has a simple answer to.
>We are staining whole-mount embryos with an antibody, and then with an
>HRP-conjugated second antibody.  We are getting signal, but there is
>some background, presumably from endogenous peroxidases.  The standard
>protocol to inactivate peroxidases is to incubate with 3% H2O2 in
>methanol, just after devitellinizing.  However, the antigen is methanol
>sensitive, so we are hand-peeling the embryos.  I've been told that
>treatment of specimens with 3% H2O2 in aqueous solutions results in
>violent bubbling and destruction of the specimen.  We will be trying a
>few things, but if anyone has a successful method for inactivating
>endogenous peroxidase without methanol, please let me know.
>
>*****************************************************************
>* Alan C. Christensen, Ph.D.          "...there are those rare  *
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>* FAX 402-472-2083                            -Sidney Brenner   *
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Not exactly a direct solution, but something you might want to bear in
mind. We stain with phalloidin for F-actin, which similarly cannot be
exposed to MeOH. Through fooling around we discovered that one can
devitellinize in Ethanol (same procedure) with a slight compromise in
morphology, and that this does not perturb phalloidin staining. You might
want to try and see a) if your antigen can withstand EtOH and then b) if
H2O2 inactivation of endogenous peroxidases works in EtOH (this I have not
done).

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i       Graham Thomas,
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