RE: Asking for optimum protocol of Whole mount in situ hybridizat ion of mouse embryo

From:

"Montague, Donna C"

William:

A couple of general points. Be sure to block endogenous alkaline phosphatase with 0.1 % levamasol (in pH 7.5 - 8.0 buffer) prior to development of the BCIP/NBT. Remember too that development of specific BCIP/NBT staining is effected by pH (8.5 - 9.0 required) and Mg2+ concentration (5mM minimum).

In particular, what are you looking for in the embryos? Is the copy number of the traget sufficient for in situ hybridization or would amplicifation be helpful? More info please.

Donna Montague, M.S.
Research Associate
Physiology/Biophysics and Orthopaedic Surgery
University of Arkansas for Medical Sciences
(501) 603-1239

-----Original Message-----
From: William Lee [mailto:wlcmwlcm@hkbn.net]
Sent: Thursday, May 23, 2002 9:19 AM
To: histonet@pathology.swmed.edu
Subject: Asking for optimum protocol of Whole mount in situ hybridization of mouse embryo

Histonetters,

I am trying to do the whole mount in situ hybridization in mouse embryos which are in different stages (7.5-10.5 dpc).

I found that there is high background after staining with BCIP/NBT. Or, there is even no expression in the embryos.

I want to ask for optimum protocol for the Whole mount in situ hybridization of mouse embryo.... Thanks a lot!!

William

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