RE: Counterstain for NBT/BCIP
Dear Oswaldo,
NBT/BCIP stained sections after ISH can be counterstained with
methylgreen indeed. Cover your sections with 100 mM Na-acetate buffer
pH 5.1 (the same buffer as your 0.1% methylgreen is dissolved) for 15
min. Than remove the acetate buffer (do not rinse) and cover sections
with the methylgreen solution for 5-10 min. Rinse briefly with tapwater
and mount aquously. Some of the methylgreen will disappear in time but
because your ISH slides were paraformaldehyde fixed before (just a
guess), a nice faint blue-green nuclear stain will stay. Do not try to
mount up your slides organically as a part of the NBT/BCIP will
disappear.
Good luck.
Chris van der Loos
Dept. of Cardiovascular Pathology
Academical Medical Center
Amsterdam - The Netherlands
Date: 4 Jul 2003 12:30:22 -0500
From: oswaldo
Subject: 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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