Counterstain for NBT/BCIP
Hi, we are doing some ISH with digoxigenin-labelled probes and an
anti-DIG-alkaline phosphatase conjugated antibody, on paraffin-embedded
sections. However, we are having trouble to find the perfect
counterstain after detection of AP activity with NBT/BCIP (very dark
bluyish/purple). It would be perfect to use methyl green because then
results might be recorded fine in B&W photos using a green filter.
However, in our hands the sections do not get the stain even after 10 or
more minutes on a warm plate (is this right or are we missing
something?). We have also tried nuclear fast red and it is OK, but
contrast is not too good in slides with heavy signal (overall
pink/purple background). In the past I also used bismark brown Y and it
gave good contrast, but the staining is very uniform and does not give
much information about the tissue or the cells present (no nuclear or
diferential staining). So the question is, any idea on a better
counterstain or on how to use methyl green in this kind of material?.
Thanks in advance,
oswaldo
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