[Histonet] Double staining with x-gal and TCR-beta

Mike Stein Barnkob mbarnkob <@t> gmail.com
Fri Mar 11 09:09:04 CST 2011

Dear Histonetters,

I am trying to do a sequential double staining against beta-galactosidase
and leukocytes in frozen mouse prostate tissue and have had little luck so
fare; when I stain for x-gal or TCR-beta alone, they show up fine, but
combined the protocols only the x-gal shows up.

What I have been doing is this:

Airdry for 5 min
Fix slides in cold acetone for 10 min
Wash slides 3x5 min in PBS
Add x-gal
Incubate overnight in humid chamber in the dark
Wash in pbs for 5 min

Incubate with blocking serum for 20 min
Wash slides 5 min in wash buffer (10 mM sodium phosphate, pH 7.5, 0.9% PBS)
Incubate with primary biotin-antibody overnight at room temperature
Wash as before
Incubate with ABC for 30 min
Wash as before
Incubate with DAB for 5 min
Wash in tap water

Any suggestions would be very welcome - is this even possible or should I
try something else?

Mike Barnkob

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