In situ with AP and Fast Red TR/Naphthol

From:Lisa C Ranford Cartwright

Hello histo folk

We are performing in situ PCR on tissue sections (fixed, embedded,
sectioned, etc). The PCR results in DIG incorporation into the DNA, which
we then detect with an anti-DIG antibody conjugated to alkaline
phosphatase. This part works fine (using a fluorescent detection system). 

We now wish to use a colorimetric substrate rather than a fluorescent one,
and FAST Red TR/Naphthol had been suggested. This is supposed to give a
bright red stain. However we also wish to counterstain with Giemsa, and I
understand the Fast Red is soluble in organics. The final methanol
concentration in the Giemsa stain will of course be low (5% stain) - is
this likely to dissolve the fast red deposits?

I've never used FAst Red TR before. Has anyone tried Fast Red TR and Giemsa
together? Any thoughts on whether it will work? Any alternatives (note we
don't want to use NBT/BCIP substrate as our tissue already as some blackish
material in it which we think will be confused with the signal).

Thanks for any suggestions, comments and assistance.

Lisa Ranford-Cartwright,Ph.D.

Division of Infection & Immunity
Institute of Biomedical & Life Sciences
Joseph Black Building
University of Glasgow
Joseph Black Building, Glasgow G12 8QQ
Scotland, UK
Tel: 00 44 141 330 2639
Fax: 00 44 141 330 4600

Lab webpages :


<< Previous Message | Next Message >>