Re: [Histonet] beta galactosidase nuclear counterstain

From:John Kiernan

The blue product of the galactosidase reaction is
an indigoid dye and it's insoluble in alcohols and
xylene. I think you must be doing something wrong
here!  Nuclear fast red isn't always the dye that
it should be. (The name has been applied to different
compounds; the one to use for nuclei is CI 60760,
made up usually as a 0.1% solution in 5% alum. Boil;
cool overnight; filter to remove crud.)

John Kiernan
London, Canada

Shaumik Adhya wrote:
> Hi Histonetters,
> I'm trying to stain myocardium for beta galactosidase (resulting in a blue
> precipitate), and have been trying to use Nuclear Fast Red as a nuclear
> counterstain - whilst this gives me reasonable nuclear staining, the process
> of dehydrating and mounting makes me lose all the beta gal.  If I use an
> aqueous mount such as gycergel, the Nuclear Fast Red dissolves out over the
> course of a week, which is less than ideal.
> My protocol for dehydrating and mounting is:
> 10 quick dips in 70% ethanol
> 2 min in 100% ethanol
> 5 min in 100% ethanol
> 5 min in Histoclear
> 2 min in Histoclear
> DPX to coverslip with.
> I'm wondering what people use as a nuclear counterstain for beta-gal, and if
> you had any tips or advice.  Thanks
> Shaumik
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