We need some double IHC for a project. As it is known, there is no possibility to combine 2 nuclear or 2 cytoplasmic Abs in classic double IHC, but only by doing it with Immunofluorescence. But is this absolute truth? Is there no possibility to combine 2 Abs of same location and to clearly see a third color???
If ayone have an experience I'll enjoy to know (there now a kit for double IHC with Ventana Benchmark, it works quite well and that's making life far more easier, but the limit is that we can only compare nuclear marker with cytoplasmic one).
Thanks everybody for your suggestions,
Wed Mar 09, 2011 9:03 am
Joined: Oct 11, 2004
Post subject: No suggestions.....
We obviously do not know how to do this/if it is possible.
I do not think it is, as you stated.
One major reason is that you are using chromogens to produce insoluble, stable ( non-interactive) pigments at the site of reaction of enzyme and its substrate.
However, I feel sure that you will find a way, philT
NB: I thought that perhaps hrn might have ideas as he is excellent in such sophisticated areas
Thu Mar 10, 2011 6:11 am
Joined: Oct 13, 2004
Post subject: double IHC
Well, I'm not sure I'll find a solution, except to do it by double IF on FFPE.
If HRN knows another way I'll be happy to learn.
By the way I thought that perhaps if both Abs are cytoplasmic, for example, but reacts with different epitopes, then in theory it could give a mixed third color, a little bit like the impressionist technique in painting...Too much empiric way of thinking I guess .
Thu Mar 10, 2011 1:05 pm
Joined: Oct 11, 2004
Post subject: I see your reasoning but...
....., the two chromogens form non-reactive, insoluble pigments ( hence the fact that they precipitate) under the conditions neccessary.
Sure a chemist could probably sort two chromogens whose final reaction products interacted with each other to form a third pigment.
Yep, IF it looks like as the light can "mix" to form that third colour.
Lets see if anyone has some sort of paradigm shift idea...
My best guess would be to use serial sections that are 3/4 microns in thickness.
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