[Zbrafish] question about peroxide block IHC/ISH

Burdine, Rebecca D via zbrafish%40net.bio.net (by rburdine from Princeton.EDU)
Thu Feb 12 14:22:19 EST 2009


Hi Serena,

Whether you need to block for endogenous AP really depends on the probe and the age of the embryos IMHO.  For some weak or finicky probes, we find blocking endogenous AP is key.  For really strong probes, there is typically no need since the signal over background is tremendously strong.

For later embryos and larvae, I think it is important because the liver will light up like gangbusters without this step.  In fact it will still light up with this step, but it is somewhat less.

Unfortunately we have had no luck getting nice specific signals with whole-mount insitus on embryos older than 7 days, but this might be our probes.  We tried sectioning after the protocol and that didn't work at all.  I'd be interested in hearing if other labs have had good luck on whole-mount ISH in larvae that are this old.

Becky

---------------------------------------------------
Rebecca D. Burdine, Ph.D.
Assistant Professor
Dept. of Molecular Biology
Princeton University
Washington Road Mof 433
Princeton, NJ 08544 
 
Phone: (609) 258-7515
Fax: (609) 258-6730
Email: rburdine from princeton.edu
Admin Assistant: Anna Schmedel (609) 258-5028


> -----Original Message-----
> From: zbrafish-bounces from oat.bio.indiana.edu [mailto:zbrafish-
> bounces from oat.bio.indiana.edu] On Behalf Of Serena
> Sent: Thursday, February 12, 2009 11:12 AM
> To: bionet-organisms-zebrafish from moderators.isc.org
> Subject: [Zbrafish] question about peroxide block IHC/ISH
> 
> I have a question about hte need for a peroxide block step in ISH. I
> have seen many whole mout protocols for IHC that require a peroxide
> block step to block endogenous peroxidases, however, the whole mount
> in situ protocols that I have seen do not have one, despite the
> similar AP detection chemistry. Can someone explain the reason for
> this? I am performing in situ on rather old embryos 10-15 days (for
> sectioning) and want to make sure I do not get a lot of background.
> 
> Also, if anyone has experience with this technique on this age range,
> could you suggest a length of time for the proteinase K step.
> 
> Thanks!
> 
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