Help with Whole Mount In-situ

john treml jftreml at
Fri Jun 6 14:52:24 EST 1997

Thanks in advance for any help.

I've been having some trouble with background in using whole mount 
in-situ hybridization with riboprobes in mouse embryos.  A light to 
dark blue background appears during the alkaline phosphatase reaction.  
The specific staining still appears darker, but the blue background 
makes for poor photographs.  I know that the background is not due to 
pooling of antibody, as I've encountered this previously.  I use a pH 
7.5 TBST buffer during immunolabelling.  I use a pH 9.5 NTMT buffer 
during the alkaline phosphatase reaction.  I filter all solutions and 
have checked the pH of my solutions.  I am wondering if this could be 
due to NTB/BCIP reagents, temperature, whatever. I doubt that the 
problem is retention of riboprobe (although I typically use 1-2 
micrograms per mL during hybridization which may be a little high).  I 
doubt that the problem is retention of antibody (as I wash 5-6 times 
1-1.5 hrs per wash; and an overnight wash).  Any suggestions about the 
possible nature of this problem or alternative protocols would be 
greatly appreciated.


Mark J. Koebbe
mkoebbe at

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