What is a good DAPI concentration for F.I.S.H. ?
mal at sanger.ac.uk
Tue May 7 04:45:40 EST 1996
The concentration of DAPI used seems to vary depending on what people
want to counterstain. If you're not wanting to see chromosome bands you
can use higher concentrations, but banding definition is usually clearer
with lower counterstain concentrations.
We use an aqueous stock at 1mg/ml, stored in the frig. After FISH, we
counterstain the chromosomes for 3-4 minutes in a 0.08mg/ml solution
(4ul stock in 50ml 2xSSC), then quickly dehydrate and mount in antifade.
We used to make up the DAPI in antifade (Citifluor AF1) but found that
the staining intensity drops off pretty quickly - maybe due to the
alkaline pH. Simple DAPI banding didn't seem to work this way either
(again maybe pH-related). I'd be interested to hear what others have to
say about this.
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