whole mount in situ hybridization

[a101anne at horus.sara.nl]

Hi Folks
I (Han Schilthuis, Hubrecht Lab, the Netherlands) am currently researching the 
effect of retinoic acid on Xenopus leavis development (one of the few left, 
I guess). In the lab we do a lot of whole mount in situ hybridization on 
embryos, and usually with good results. This time, however, we are at a loss. 
We got a plasmid construct from another group, after they had published its 
pattern of expression in  Xenopus embryos as studied with the above method. 
I re-constructed the probe they had used, thoroughly checked it, and tried 
using it as a template for RNA probe transcription using a transcription kit 
(mESSAGE mACHINE from Ambion). We did get some (DIG-labeled) synthetic RNA, 
but no signal whatsoever. I checked the template (sequenced it, even), used 
the same protocol as in the  original paper, even used their construct, 
fiddled a bit with the temperature, still no luck. I know this is rather a 
classical example of 'I can't make it work while I do everything by the book' 
but I am rather desperate for it to work as it looks like being rather crucial 
for the rest of my research. If anybody out there has anything (potentially) 
useful (however remote) to contribute, I would surely be very grateful.
Looking forward to any reactions,  

regards, HAN


Dr Johannes G. Schilthuis
Hubrecht Laboratory
Netherlands Institute for Developmental Biology
Uppsalalaan 8
3584 CT Utrecht NL

phone +31 30 2510211     fax: +31 30 2516464    Email han at hl99.hubrecht.nl