RE: [Histonet] Re: Two questions about double IHC

From:"Turner, Scott"

I think there might be a way to do this.  The tyramide doesn't actually
bind to the immune complex but rather precipitates and covalently binds
to the tyrosine residues in the tissue proteins.  Therefore, I think you
could use the non-biotin fluorescyl-tyramide kit to stain your first
antibody, then elute the immune complex leaving the fluorescyl-tyramide
behind.  Then you'd have to get a little tricky.  Since both of your
antibodies are rat anti-mouse, you'll have to pre-label your other
primary somehow.  The easiest thing to do would be to biotinylate that
primary.  Alternatively, you could pre-complex your biotinylated
secondary Ab with your second primary and block the unbound secondary Ab
with mouse serum so that it doesn't stick to your first primary
antibody.  You can figure out the concentrations that work by yourself
or you could use the biotinylation and blocking reagents from the Dako
ARK.  After you've done that, you can put the primary/secondary complex
on the tissue and follow with your biotin-based TSA and a
Streptavidin-Texas red.

Anyway, that's what I could come up with.  I know it sounds really
complicated but I think it might work (with a LOT of trial and error to
get it right).  Does anyone out there have any input on this?

Scott Turner
Schering-Plough Biopharma
(formerly DNAX Research Institute)
Palo Alto, CA

-----Original Message-----
[] On Behalf Of Johnson,
Sent: Tuesday, April 24, 2007 11:55 AM
Subject: [Histonet] Re: Two questions about double IHC

Amy, you asked:
I need to do double IHC on FFPE mouse tissue. Unluckily two antibodies I
have to use are all rat anti-mouse. The protocol for these antibodies
are very similar: Pro K digestion, Primary antibody, Rb anti-Rat 2ND
antibody and the rest is following PerkinElmer TSA biotin system
instruction (Streptavidin-HRP, biotinylated-tyramide and
   I will perform the two IHC sequentially. I have two questions:
  1. Do I need to do Proteinase K for the second antigen?
  2. Can I keep all other steps the same and just use Streptavidin-FITC
or Streptavidin-Texas Red at the final step to differentiate two
  BTW, could you recommend a good website to learn double IHC?

You cannot do the double staining as you describe. By using the same
detection method for both, you will end up labeling both antibodies with
both colors. If you try eluting the first antigen-antibody complex after
labeling with the first antibody, you will lose your first label and
will only show labeling of the second. (does this make sense?)

It's best if you have two different animal species, or use two different
methods for detection. Having both from the same species AND having both
require tyramide amplification makes your task next to impossible by

It may be possible to do this chromogenically using DAB in a sequential
staining protocol, but it's going to be complicated.

It's even more complex if you think you will have co-localization.

I'll take a stab at it putting something together that might work, but
I'm hoping Chris van der Loos (if he's on list) or Gayle Callis comes up
with something easier:

Primary antibody #1 labeling:
Pro K digestion, rat anti-mouse primary Ab 1, biotinylated Rabbit
anti-rat, Streptavidin-HRP, biotinylated-tyramide, Streptavidin-HRP,
DAB. Elute using heat-induced antigen retrieval (shouldn't need PK
digestion at this point) Rat anti-mouse secondary Ab 2,  HRP-labeled
anti-rat antibody, FITC-tyramide, anti-FITC AP, NBT/BCIP or other stable
chromogen Nuclear fast red counterstain (If the above chromogens used).

Co-localization will be impossible to see with this color combination.

You will need adequate controls doing this experiment. That could
potentially be a lot of slides with one experiment.

It might be easier to try to optimize one of your antibodies with a
higher concentration and no tyramide. Have you tried not using tyramide?
Sometimes using polymer HRP systems work well. Look for anti-rat
polymer, I think Biocare Medical might have some?

Or replace one of the antibodies with one made in a different species
(should be much easier), and then try to optimize it without tyramide.
Or use labeled antibodies, one with biotin, one with FITC, and use two
different detections.

You're going to have to simplify this before you can really get good
results that you can trust.

Best reference I know is Chris van der Loos' book on Multiple
Immunostaining, ISBN 1-85996-187-8.

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110

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