I have used an anti-rabbit primary antibody (X) on first day, and another anti-rabbit primary antibody (Y) on 2nd day to observe co-localization. Is it possible to perform this method ? What is the chance of false positive interaction? Will reviewer mind in this issue?
Thanks !!
To detect two proteins on the same slide, you need two secondary antibodies with different fluorescent emissions (resulting in two different colors, e.g. green and red). But this is only possible if you have different species of primary antibodies, because otherwise there is a complete crossreaction.
Or maybe I'm not getting your question right. How do you plan to check for interaction of your two proteins?
Yes , I am using FITC and TRITC which gives different fluorescence. But the problem is, both the primary antibodies are raised with same species (i.e. rabbit).
ok, so then how are you planning to distinguish between your two proteins? Because both primary antibodies will react with both secondary antibodies.
Yes, that is the thing i am wondering for. Is there will be the reaction if i wash with PBS or TBST before labeling 2nd antibody?
I think it will be difficult to be sure there's no crossreaction.
You might read this attached publication for a possible protocol for a double staining. But I don't think you can trust the results from "normal" double staining with anitbodies from the same species.
http://www.ncbi.nlm.nih.gov/pubmed/8918908
Try to conjugate your primary antibodies with different fluorochromes directly. Do not use secondary antibodies. And perform control immunofluorescences with only one fluorochrome-conjugated primary antibody to see whether they're working as your primary with a fluorochrome-conjugated secondary
Try using a tyramide signal amplification detection method.
In brief, the first primary antibody is incubated with a HRP labelled secondary (anti-rabbit), then a fluorochrome (e.g. FITC) labelled tyramide visualisation reagent is applied. The oxidised labelled tyramide becomes covalently bound to the target tissue which can then allow the primary antibody to be eluted off the tissue (generally using a heat elution via heat induced epitope retrieval (HIER)).
Once this primary antibody is eluted from the tissue you can apply a second primary of the same species as the first primary.
I use Perkin-Elmer's OPAL reagents quite successfully, they have a base 4 colour kit which allows the visualisation of 3 separate primary antibodies regardless of the primary species (this can be expanded up to a 7 colour kit)
http://www.perkinelmer.com/product/opal-4-color-ihc-kit-50-slides-nel794001kt
Thank you everybody for the valuable suggestions. I will try to get 1st or 2nd primary antibody raised with different species.
try hybridization technique: use your primary antibody x, add secondary antibody for X lets say FITC. then block your samples again. then only use your second primary ab y, followed by next sec antibody in TRITC for example. It is possible to use two antibody raised in same speicies but you cannot use them together in one pipetting.
If you see some single positive cells then your abs are not cross reacting. If you see all double positive, then your primary antibodies are cross reacting with each other and yoou need to change one of the abs.
Good Luck.
Excellent suggestions so far and I am sure you all will find this webinar helpful for more insights into dealing with this issue: https://www.rndsystems.com/multi-color-fluorescence-ihc-using-primary-antibodies-raised-same-host-species
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