[Histonet] Re: Two questions about double IHC

From:"Johnson, Teri"

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,
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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