Post subject: Getting rid of NBT/BCIP precipitate from wax sections
I have some paraffin sections that I've used for immunohistochemistry (using an alkaline phosphatase-conjugated secondary antibody and NBT/BCIP as a substrate), and coverslipped with an aqueous mounting medium (Fluoromount G). I would now like to de-coverslip and re-stain these sections with a different histochemical protocol (i.e. Masson's trichrome or H&E). Does anybody know if there is a way to "clear away" the NBT/BCIP precipitate from my previous IHC?
Any advice would be greatly appreciated.
Fri Jan 06, 2012 3:55 pm
Joined: Oct 11, 2004
Post subject: Hi Andrew
Don't bother to remove!
Just do your H&E over the BCIP/NBT.
Sure, if this is not appropriate for your needs/does not work very well.....
I have not tried to remove this pigment but I have sometimes noted that when I have dehydrated only SOME NISH sections ( probe detected using NBT/BCIP) the pigment would dissolve out in dehydrating alcohol) so, have 3 suggestions that I list in increasing harshness...( eg: use a Coplin jar)
1. Place in 100% alcohol
2. Place in 1%HCL in 70% alcohol ( Haemalum differentiating solution)
3. Place in 1% household bleach.
Agitate gently and rinse in tapwater after an initial say, 30secs, and examine microscopically to see if there is any effect. If, after 5mins , there is no difference in 1, move to 2....etc.
Please let us know which, if any, works and if subsequent tinctorial staining is successful.
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