[Histonet] RE: beta galactosidase nuclear counterstain

From:"C.M. van der Loos"

Using X-gal as substrate and ferri/ferro-cyanide as chromogen for b-galactosidase activity you obtain a turquoise/blue reaction product that survives everything: alcohol, xylene, cooking in citrate, in situ procedure.....

Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center
Amsterdam - The Netherlands

---- Original Message ----- 
>From  Shaumik Adhya  
Date  Fri, 23 Apr 2004 10:46:59 +0100 
To  histonet@lists.utsouthwestern.edu 
Subject  [Histonet] beta galactosidase nuclear counterstain 

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

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