Transgenic Alk-Phos problems

Peter Ashby p-ashby at nimr.mrc.ac.uk
Fri Jul 25 15:43:51 EST 1997


Hi

I have generated a mouse transgenic line with Alk-phos as reporter.  I am
analysing in whole 8-12day old embryos.  The problem I have is relatively
high background.  This is higher than we routinely get in the lab with
wholemount in situs.  Staining is pH8.8, 1mg/ml NBT, 0.1mg/ml BCIP (x-phos).
Fixation is 30min in 4% paraformaldehyde/PBS, wash, heat 65C for 45min; 
2-4hr in 0.24mg/ml levamisole, then stain.  I now include levamisole in
stain solution which helps.

I would be grateful for advice on how to bring the background down. 
Background begins to come up in about 15min, specific stain about 30min.  I
have a line (just, yahoo!) so can get lots to experiment on.

TIA

Peter
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Dr. Peter Ashby                          National Institute for Medical Research
Eukaryotic Molecular Genetics           London, England
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