Re: [Histonet] granules after X-gal staining

From:John Kiernan

Beta galactosidase is present in normal cells but, like other lysosomal enzymes, it is active at low pH (4 to 5.5). The bacterial enzyme, whose gene is introduced into cells by infection or transfection, has a higher pH optimum (7.5). With the indigogenic medium at this higher pH the lysosomal enzyme should not show up unless the incubation is unduly prolonged. The bacterial enzyme is also used as a label for secondary antibodies in multi-colour imunohistochemistry. As you suggest, a "normal" tissue without the bacterial enzyme would be a sensible negative control.

See Biotech. Histochem. 82(2):73-103 (2007) for a review of X-gal and other indigogenic substrates. (This whole issue of the journal is about indigo and related dyes; it makes for some interesting reading that goes beyond the field of histotechnology.)

John A. Kiernan
Dept of Anatomy & Cell Biology        
The University of Western Ontario            
LONDON,  Canada  N6A 5C1
= = =
----- Original Message -----
Date: Friday, April 4, 2008 0:18
Subject: Re: [Histonet] granules after X-gal staining
To: Yves Heremans ,

> 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  
> > 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 
> > 
> > 
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> > 
> > 
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