RE: Counterstain for NBT/BCIP

From:"C.M. van der Loos"

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 
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 
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,

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