In situ hyb with Digoxigenin

Dr. Kent L. Nastiuk nastiuk at
Tue Feb 6 13:14:20 EST 1996

In article <4f71vi$20dg at>,
   ksayani at (Karim Sayani) wrote:

>I have previously conducted in situ hybridization using riboprobes 
>labeled via in vitro transcription with Digoxigenin-UTP as the label.
>However, upon switching to a new probe, both sense and antisense probes
>give me positive staining.  I looking in a cell culture system where I 
>confident that the target desired exists in abundant quantities.
>Does anyone out there have any ideas?  I have played around 
>with the stringency of the washes.  I either get the staining with both 
>probes or it all disappears.
first, while it is important to wash stringently, it is equally 
important to eliminate non-specific binding in the first place.  have 
you tried raising your hybridization stringency in conjuction with 
increasing the wash stringency?

second, you might also try RNAse treatment during the washes.

good luck


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