I'm now trying to do double IHC staining on newborn rats brain sections. I have tried several times. It seems that it works well when the nucleus is to stained, such as BrdU/NeuN, but I always fail when the cytoplasm is to stained, such as BrdU/GFAP, BrdU/DCX or BrdU/vimentin. Is it because of the 2HCl I used for DNA denaturation?
Here is my protocol:
1, deparadfinization
2, pressure cooker (2 min) with antigen unmasking solution
3, 37℃ 2NHCl 30 min
4, blocking 1h
5, 1st antibody overnight
6, 2nd antibody 1h
Did you try double immunofluorescence or double immunoperoxidase? You may read
Article Characterization of the "sporadically lurking HAP1-immunorea...
Do your cytoplasmic antibodies (GFAP, DCX, vimentin) show realiable staining patterns in your tissue slides when applied as single antibody (no double IHC)? If yes, try to apply the cytoplasmic antibody first and the nuclear antibody as 2nd primary antibody.
Before adding the 2nd primary antibody you should add microwave treatment (e.g. 7x5 min) to denature bound antibody
molecules and blocking cross-reactivity between sequential rounds of staining.
Hello, Dr. Beschorner,
Thank you for your answer.
What I'm doing now is to do antigen retrieval respectively first, (pressure cooker and HCl for GFAP and BrdU respectively) , then add the mixed primary antibodies, either BrdU/GFAP or BrdU/VI.
So you recommend me to do antigen retrieval and primary antibody separately, right? I assume it will be like followings,
1, deparafinization
2, pressure cooker (or microwave)
3, primary antibody for GFAP, DCX or VI ( cytoplasmic antibody )
4, HCl
5, primary antibody for BrdU (nuclear antibody)
6, mixed second antibodies
Please let me know if there is any misunderstanding. Thank you for your time.
yes, you should apply the primary antibodies in 2 steps. And you need 2 different chromogenes for each primary antibody (e.g. DAB + Fast Red). You may also see ref. PMID: 7822770 (Lan et al 1995).
For use of non-biotinylated primary antibodies:
1, deparafinization
2, pressure cooker (or microwave)
3, block endogeneous peroxidase (1% hydrogen peroxide)
4, block non-specific binding sites with normal swine serum
5, 1st primary antibody (e.g. GFAP)
6, visualize binding of 1st primary antibody with an biotinylated secondary antibody directed against the species of the 1st primary antibody,
streptavidin and biotinylated horseradish peroxidase, chromogene (e.g. DAB)
7, second microwave treatment
8, 2nd primary antibody (e.g. BrdU)
9, visualize binding of 2nd primary antibody with an biotinylated secondary antibody directed against the species of the 2nd primary antibody,
streptavidin and biotinylated horseradish peroxidase, chromogene (has to be different than with the 1st antibody, e.g. Fast Red, Fast Blue,...)
In which order your antibodies will give the best results (cytoplasmic or nuclear first?) may differ with different combinations of antibodies, just try variations if you are not satisfied with the staining.
Hi Yun,
My lab, NDB bio, has experience with these kinds of stains on rat brain as well as human tissue. If you want, you can send an email to [email protected] to get some advice. You can also take a look at www.ndbbio.com for examples of some of the stains.
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