Beat Gal Re: [Histonet] Blood smear

From:Gayle Callis

A very reliable Beta Gal Staining kit can be purchased from Roche ( 
1 828 673).  For proper staining, there are fixation requirements to 
optimize staining but a good positive control (we used cells transfected 
with a B galactosidaase encoding construct) as it is really necessary to 
know if kit i.e. staining is working correctly.  The kit insert is highly 
informative and probably found on Roch website.

>Problem 2. No blue product.
>The simplest explanation is that no blood cells
>contain a beta-galactosidase. Have you done the
>method with a similarly prepared film of blood (or
>other cells) known to give a positive reaction for
>Which enzyme are you after?
>Are you staining for endogenous beta-galactosidase
>(a lysosomal enzyme that works at pH~5) or for the
>bacterial lac-Z enzyme (pH~7+)? The lac-Z gene is
>often coupled to more interesting genes in
>transfection experiments etc. Cells that express
>the interesting gene also make a
>beta-galactosidase that can generate an insoluble
>blue pigment from X-gal at pH>7.
>John Kiernan
>Anatomy, UWO
>London, Canada
>"Xing, Zhiqing" wrote:
> >
> > I am trying to do X-gal staining on mouse blood smears. But the blood
> > film keeps coming off the slide. I believe the smear is thin enough.
> > After making the smear, I dried it overnight at RT or 2-3 hours in
> > vacuum. Then if I fix it with 100% methanol, the slide would be OK, the
> > blood doesn't come off during the staining, but I don't get any X-gal
> > stain. I tried fixation with acetone for 20 min, the blood stayed on the
> > slide when in acetone, but it came off when transfered to staining
> > solution. I also tried fixation with glutaraldehyde or PFA, the blood
> > just comes off even in the fixative solutions. In the begining, I was
> > using Superfrost plus slides. Somebody on the histonet said the blood
> > smear might not like the plus slides. So I changed to the regular
> > slides, but the problem remains.
> > Can anyone give me some suggestions? Thanks.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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