More on In situ with AP and Fast Red TR/Naphthol

From:Lisa C Ranford Cartwright

Dear histo-netters 

Thanks for the very useful and rapid responses! I have now more ideas,
suppliers, etc.

The reason we don't want to use NBT/BCIP is that the deposit looks like
malaria pigment, which we have a lot of in these sections. Previously we
have used fluorescence, but we are having trouble with autofluorescence in
this batch of tissue, particularly on the red cells - and we are amplifying
malaria parasites within these red cells. We have tried various things
(cupric sulphate, Sudan Black) to get rid of the red cell autofluorescence
but these haven't worked. 
Any other suggestions for getting rid of this gratefully received!

The tissues (placentae) were fixed in paraformaldehyde, embedded in
paraffin and sectioned. This was done elsewhere by somebody else, and it
can't be repeated. We have deparaffinised etc here. For future reference,
is the view of the group that paraformaldhyde/paraffin will (always) give
autofluorescence, or is it possible to avoid this?  

We're going to give the FastRed a go (as we have the reagents) and see what
happens. I'll report the findings on the Giemsa counterstaining to the
histonet group.
We did consider sensitivity with Fast Red but as we are PCR amplifying the
target there should be lots of it there. Mounting in aqueous mountants is
fine for us - we don't need to keep these slides after reading. 

Thanks again for the 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

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