DIG in situs on late embryos

[Frances Hannan Zoology]

Dear Drosopholists,

I am looking for a DIG in situ method which will work on late
Stage 17 embryos (ie after cuticle formation). I have tried several
variations of the basic Tautz & Pfeifle protocol without much luck
so far. These protocols work fine in my hands using early embryos 
with other probes. Unfortunately the genes I am interested in are 
expressed in the central nervous system and no mRNA is seen on 
Northerns prior to 15 hours of development. My probes seem OK 
(ie 300-400 bp long, 10-100ng/ul) but all give very low signal
/high background. I have tried random priming, single stranded PCR 
and riboprobe labelling. I suspect either my probes or the alk phos 
stain aren't getting in through the cuticle. 

Alternatively I could go to sections if anyone has a DIG method 
that will work on sectioned material. I have done S35 in situs on 
late embryos but I am not currently set up to do this and would 
prefer to get this DIG thing working since the localizations are 
so much better.

Thanks in advance for any/all suggestions, 

Frances Hannan
AFRC LMS, Zoology, Cambridge, UK
flh at mole.bio.cam.ac.uk