[Histonet] combined ISH/IHC

[Mikael Niku]

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 DAB precipitate).
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
quantitation).

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 detection).

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
protocol.

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

- Mitäkö mieltä olen länsimaisesta sivistyksestä?
  Minusta se olisi erinomainen ajatus!
					 (Gandhi)



> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu 
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf 
> Of Tora Bardal
> Sent: 28. tammikuuta 2004 17:16
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] combined ISH/IHC
> 
> 
> Hello
> Does anyone have a working protocol they can share?
> 
> I'm trying to combine in situ hybridization (RNAprobe) and 
> immunohistochemistry (PCNA) on fish paraffin sections.
> So far: ISH ok, IHC doesn't work, or ISH no signal, IHC ok. 
> PCNA(DAKO) 
> needs AR.
> 
> Before spending more time an money I would like to have some 
> good advices 
> on what to do when, ab simultaneous incubation? stepwise? 
> crossreactions? 
> blocking ?
> I'm using sheep anti-DIG-AP/BCIP/NBT (ISH) and DAKO Envision 
> HRP (mouse)/DAB.
> 
> Tora
> 
> 
> Tora Bardal
> Department of Biology,
> Norwegian University of Science and Technology (NTNU)
> Brattøra Research Center
> N-7491 Trondheim
> Norway                                   + (47)73 59 09 38 / 970 25256
> E-mail: Tora.Bardal <@t> bio.ntnu.no         fax:+ (47)73 59 63 11 
> 
> 
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