[Histonet] combined ISH/IHC

[bjdewe <@t> aol.com]

I would love a copy of this protocol! Sorry to bother the list but I couldn't find the email for Mikael ;-)

Cheers,
Lorie


Live as if you were to die tomorrow. 
Learn as if you were to live forever. - Gandhi -




-----Original Message-----
From: Jimmy Lao <lao_ji <@t> yahoo.com>
To: histonet <@t> lists.utsouthwestern.edu
Sent: Wed, 6 Jun 2007 6:03 am
Subject: [Histonet] combined ISH/IHC



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

ear Tora,
we are routinely doing combined ISH + IHC. The ISH is
or genomic DNA,
o with a RNA target this might be slightly more
ricky. But RNA in
issues is surprisingly well preserved, so I think
his should work.
I spent a LOT of time trying to find a useful protocol
or a double
taining. The only generally useful way I know is to
se the tyramide
ignal amplification system. 
This is how it goes:
1) Start with IHC: antigen retrieval, primary
ntibody, biotinylated /
RP-conjugated secondary antibody, and then the
yramide reaction. Now
ou have a covalently bound label (biotinylated
yramide, for example)
n the tissue, so you don't need to worry about
ntigen destruction or
tripping of bound antibodies.
2) Then perform the whole ISH procedure.
3) After you have completed the NBT/BCIP color
eaction of ISH, finish
he IHC (if using biotinylated tyramide, add
RP-avidin, then DAB).
This protocol allows you to begin with IHC (to avoid
estruction of
ntigens in the harsh ISH treatments) but to do the
isualization step
nly after ISH (to avoid false negatives caused by the
AB 
recipitate).
s additional bonus, you get signal amplification for
HC, and a nicely
ven DAB color intensity (nice provided that you don't
eed any idea of
uantitation).
If it's the RNA that is the more sensitive target, I
uess you could do
his the other way round (to use tyramide for RNA
etection).
The only drawbacks I can think of are:
1) A few additional incubations due to the tyramide
rotocol (these are
uick to do, though)
) The commercial tyramide reagents (which you need to
se at least if
ou're doing any commercial stuff, as the technology
s patented) are
retty expensive, if you're doing lots of slides.
Please let me know if you would like to get a copy of
ur exact
rotocol.
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
  Mikael Niku       
  University of Helsinki, Dept. Basic Veterinary
ciences
  URL: www.helsinki.fi/~mniku/
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
- Mitäkö mieltä olen länsimaisesta sivistyksestä?
 Minusta se olisi erinomainen ajatus!
                    (Gandhi)

       
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