[Histonet] X-gal staining

[John Kiernan]

Dear ndevans <@t> stanford.edu
 
You may be detecting endogenous animal in addition to bacterial (lac-z) beta-galactosidase activity. Indigogenic methods were recently reviewed in Biotechnic & Histochemistry 82: 72-103 (2007). The pH of the indigogenic substrate solution is important. Many other factors can cause false positive and false negative X-gal staining.  Do your homework!
 
John Kiernan
Anatomy, UWO
London, Canada
= = =
----- Original Message -----
From: Nicholas David Evans <ndevans <@t> stanford.edu>
Date: Thursday, August 13, 2009 15:51
Subject: [Histonet] X-gal staining
To: histonet <@t> lists.utsouthwestern.edu

> Hello,
> 
> I was wondering whether anyone might advise me on X-gal 
> staining. 
> 
> I am looking at the expression of LacZ knocked into the locus of 
> our gene
> of interest. We cryoembed, take sections and stain for beta-
> galactosidaseactivity using X-gal. The blue stain is often 
> punctuate and noticeably
> limited to the nucleus, whereas in other areas the staining is 
> diffuse and
> appears throughout the cytoplasm of the cells. I have guessed 
> that this
> may be related to the level of expression; i.e. that in cells 
> with a low
> reporter activity beta-gal is limited to the nucleus, but that 
> at higher
> expression the whole cell becomes riddled with it. If anyone knows
> anything about the subcellular distribution/localisation of beta-
> gal in
> mammalian cells, or can direct me to some good literature I 
> would be
> grateful.
> 
> Best wishes
> Nick
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