[Histonet] combined ISH/IHC

From:Jimmy Lao

Dr. Mikael Niku,
I kept this email since 2004, because I thought it
might be someday useful for me ( see below). now I
really want to try these combined ISH/IHC, can you
send me a protocol? 
By the way, I check your web page, it is very
interesting, I like it. Hope you are still in
histonelist, so you can help me.
I don't have experience in Tyramide reaction. Somebody
in our lab used to try PerkinElmer's, but it seems not
I like tyramide biotinylated, I think then ABC kit +
DAB will get strong amplifying signal. Can anybody
here share with me some tyramide with DAB staining
experience and suggest the vendor you are using?  
Thank you very much!

Dear Tora,

we are routinely doing combined ISH + IHC. The ISH is
for genomic DNA,
so with a RNA target this might be slightly more
tricky. But RNA in
tissues is surprisingly well preserved, so I think
this should work.

I spent a LOT of time trying to find a useful protocol
for a double
staining. The only generally useful way I know is to
use the tyramide
signal amplification system. 

This is how it goes:

1) Start with IHC: antigen retrieval, primary
antibody, biotinylated /
HRP-conjugated secondary antibody, and then the
tyramide reaction. Now
you have a covalently bound label (biotinylated
tyramide, for example)
in the tissue, so you don't need to worry about
antigen destruction or
stripping of bound antibodies.

2) Then perform the whole ISH procedure.

3) After you have completed the NBT/BCIP color
reaction of ISH, finish
the IHC (if using biotinylated tyramide, add
HRP-avidin, then DAB).

This protocol allows you to begin with IHC (to avoid
destruction of
antigens in the harsh ISH treatments) but to do the
visualization step
only after ISH (to avoid false negatives caused by the
As additional bonus, you get signal amplification for
IHC, and a nicely
even DAB color intensity (nice provided that you don't
need any idea of

If it's the RNA that is the more sensitive target, I
guess you could do
this the other way round (to use tyramide for RNA

The only drawbacks I can think of are:

1) A few additional incubations due to the tyramide
protocol (these are
quick to do, though)
2) The commercial tyramide reagents (which you need to
use at least if
you're doing any commercial stuff, as the technology
is patented) are
pretty expensive, if you're doing lots of slides.

Please let me know if you would like to get a copy of
our exact

+  Mikael Niku		 
+  University of Helsinki, Dept. Basic Veterinary
+  URL: www.helsinki.fi/~mniku/

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