[Histonet] granules after X-gal staining

[Houston, Ronald]

Yves,

You don't say whether or not you are briefly fixing the sections. The
binding of the b-galactosidase to lysosomes is relatively loose, and
that may contribute to what you are seeing. Brief fixation in
paraformaldehyde vapor may suffice.
Also, something worth considering, have you tried inhibiting the enzyme
with e.g. D-galactonolactone, lactose and/or p-chloromercuribenzoate?

This is only speculation as I have never come across this phenomenon
personally, but, as with Ray, have never performed the technique on
pancreas.

One way to get by the problem of diffusion artefact, if that is what
this is, would be to employ a semi-permeable membrane enzyme
histochemical technique.


Ronnie Houston, MS, HT(ASCP)QIHC
Anatomic Pathology Manager
Nationwide Children's Hospital
700 Children's Drive
Columbus, OH 43205
(614) 722 5465
Ronald.Houston <@t> NationwideChildrens.org
Columbus Children's Hospital is now Nationwide Children's Hospital
www.NationwideChildrens.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rachel,
Rivka (NIH/NEI) [E]
Sent: Friday, April 04, 2008 8:25 AM
To: koellingr <@t> comcast.net; Yves Heremans;
histonet <@t> lists.utsouthwestern.edu
Cc: Smith, Roberta (SAIC)
Subject: RE: [Histonet] granules after X-gal staining

We used to see this all the time and couldn't figure out the problem.
The histologist even tried filtering all the solutions used for
perfusion, staining solutions and many other things but never could
determine the cause.  Any suggestions?


-----Original Message-----
From: koellingr <@t> comcast.net [mailto:koellingr <@t> comcast.net]
Sent: Fri 4/4/2008 12:16 AM
To: Yves Heremans; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] granules after X-gal staining
 
I haven't seen any replies and I am scientifically curious.  The
beta-galactosidase is simply the enzyme that splits x-gal to eventually
produce that classical blue chromogen deposit.  The acinar cells of
pancreas are laden with known ..ase's (lipoxygenase, proteases, amylase,
lipase, elastase, tryptase, etc, etc ase's) and probably unidentified
ones.  Is it possible that some promiscuous enzyme is substituting
enzymatically for beta-galactosidase to get your staining of tiny round
blue granules in cytoplasm.  If you are working in frozens, your enzymes
could all be very active.  Have looked at lots of x-gal staining but
never in pancreas.  Have you stained a normal, not b-gal expressing,
mouse pancreas? Am curious and hope someone has done this.

Ray Koelling
PhenoPath Labs
Seattle, WA

-------------- Original message -------------- 
From: Yves Heremans <Yves.Heremans <@t> vub.ac.be> 

> Dear Histonetters, 
> 
> Does anyone know why I am getting granules (tiny, round blue granules 
> in the cytoplasm) after X-gal staining on frozen sections of mouse 
> pancreas ? 
> 
> Regards, 
> 
> Yves 
> 
> 
> _______________________________________________ 
> Histonet mailing list 
> Histonet <@t> lists.utsouthwestern.edu 
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