RE: [Histonet] granules after X-gal staining

From:"Houston, Ronald"


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
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
=0Ahistochemical technique.

Ronnie Houston, MS, HT(ASCP)QIHC
Anatomic Pathology Manager
Nationwide Children's Hospital
=0A700 Children's Drive
Columbus, OH 43205
(614) 722 5465
Columbus Children's Hospital is now Nationwide Children's Hospital
-----Original Message-----
[mailto:histonet-bounces@lists.utsouthwestern=2Eedu] On Behalf Of Rachel,
Rivka (NIH/NEI) [E]
Sent: Friday, April 04, 2008 8:25 AM
To:; Yves Heremans;
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: []
Sent: Fri 4/4/2008 12:16 AM
To: Yves Heremans; histonet@lists.utsouthwestern=2Eedu
Subject: Re: [Histonet] granules after X-gal staining
=0AI haven't seen any replies and I am scientifically curious.  The
beta-galactosidase is simply the enzyme that splits x-gal to eventually
=0Aproduce that classical blue chromogen deposit.  The acinar cells of
=0Apancreas 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=2E

Ray Koelling
PhenoPath Labs
Seattle, WA

=0A-------------- Original message -------------- 
From: Yves Heremans  

> Dear Histonetters, 
> =0D> 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 mailing list

Histonet mailing list
----------------------------------------- Confidentiality Notice:=0AThe following mail message, including any attachments, is for thesole use of the intended recipient(s) and may contain confidentialand privileged information. The recipient is responsible tomaintain the confidentiality of this information and to use theinformation only for authorized purposes. If you are not theintended recipient (or authorized to receive information for theintended recipient), you are hereby notified that any review, use,disclosure, distribution, copying, printing, or action taken inreliance on the contents of this e-mail is strictly prohibited=2E Ifyou have received this communication in error, please notify usimmediately by reply e-mail and destroy all copies of the originalmessage. Thank you.

Histonet mailing list

<< Previous Message | Next Message >>