Dear All,
I tried to study co-localization of two different proteins by using immunofluorescence staining, since the western blot result of both protein shows significantly increase in nuclear extracts. However, both protein antibodies are rabbit host species, and I couldn't find other species antibody which has the same epiptope in both protein. I am afraid that changing the antibody can result different outcome.
Is there a way double-color immunofluorescence staining for two different protein, but have same rabbit host species antibodies?
Thank you very much
Cheerios,
Hi, this topic has been discussed on researchgate and in many other forums. To start with, you can follow this protocol,
http://www.ihcworld.com/_protocols/general_IHC/immunofl_double_squential.htm
You may want to include control sections with only one primary antibody and two secondary antibodies. This is to check whether your 2nd secondary antibody reacts with 1st primary antibody.
Good luck.
Dear Asa Wu,
So, your primary antibodies are both raised in rabbit...Are they conjugated?
I'm not sure what species you are targeting, but, if you require a secondary antibody, then you'll have an issue. Both your secondaries have to be anti-rabbit and they will most probably cross-react.
This paper describes the technique for two mouse antibodies but it should be the same with rabbit.
http://jhc.sagepub.com/content/41/8/1273
Hi Asa,
If you can find primary antibodies that are different isotypes (i.e. IgG, IgM), you may use corresponding secondary (i.e. anti-IgG, anti-IgM) without having cross-reaction.
Also, there are antibody conjugation kits commercially available, you can label you primary antibodies with different colors, and there is no need for secondary antibodies. The downside is that those kits are usually costly (~at the price of a new primary antibody).
Alternatively you can try the Fab fragments like Víctor suggests, we have successful experience with this method, here is another illustration of how it works,
https://www.jacksonimmuno.com/technical/products/protocols/double-labeling-same-species-primary
Good luck!
If they are already conjugated, then you don't need to worry about secondary-Abs - which is the most tricky issue with species cross-reactions.
So, assuming the Abs you use are good ones (as in; have good specificity), then it should work. Just make sure to use blocking solution for 1 hour.
To make sure prepare 4 slides; an unstained one, a stained with Ab 1 alone, one with Ab 2 alone and one with Ab1+Ab2, to make sure that it's accurate.
Hi Asa,
You can try to visualise the first primary with a tyramide signal amplification (TSA) method. This will covalently bind a fluorochrome labelled tyramide at the site of the primary antibody. Once this is bound the first primary can be heat eluted from the tissue and the second primary antibody added then visualised by any method you wish.
I use the opal staining system from perking-elmer, but most TSA systems will work. (Note: TSA is very sensitive, you will need to dilute your primary antibody)
this is the link to the kit I use:
http://www.perkinelmer.com/product/opal-4-color-ihc-kit-50-slides-nel794001kt
Cheers
Dear Asa,
As suggested above, I think the easiest solution would be to label your primary antibodies with different fluorochrome prior to the incubation step with the cells. Another alternative which I have found very useful to study protein co-localisation is to use a Proximity Ligation Assay (sold by Sigma inder the name DUOLINK). Here is the link :
http://www.sigmaaldrich.com/life-science/molecular-biology/molecular-biology-products.html?TablePage=112232138
The principle is the same as a normal immunofluorescence but your secondary antibodies are respectively linked to a PLUS and a MINUS oligonucleotide sequence which is amplified by a polymerase only if your 2 proteins are interacting, resulting in a fluorescent signal. This does not solve your problem of having 2 rabbit primary antibodies, but again, the primary antibodies can be coupled to the oligonucleotide sequences directly, rending the use of a secondary antibody unnecessary. The procedure is listed in the FAQ.
Good luck with your experiment, whichever way you do it.
Best,
Damien
Hello Asa,
You can use 2 antibodies raised in same species. Use your first primary ab incubate with first secondary ab. then rinse properly your material (tissue:cryo or paraffin/cells: adherent or suspension). block your samples again, apply second primary ab, followed by second secondary ab. just make sure your fluorochromes are far apart (FITC and TRITC for example). if you see some single positive cells in both fluorochromes, you abs are working fine, you may go on with your experiment. If you see all singals colocalizing, then your abs are cross reacting with each other and you might consider to change one of the antibody, find another speicies.
In short, it is possible to perform double immunofluorescence with abs raised in same speicies.
Good Luck!
Hello Asa,
if you want to perform a double staining with two primary antibody raised in the same host species you can follow the paraformaldehyde vapour blocking method originally proposed by Wang & Larsson (1985). Our lab had succesfully employed it years ago and I used it recently (for more details see the reference: C. Salio, L. Lossi, F. Ferrini and A. Merighi (2005) Ultrastructural evidence for a pre- and postsynaptic localization of full-length trkB receptors in substantia gelatinosa (lamina II) of rat and mouse spinal cord. European Journal of Neuroscience, 22:1951-1966.).
Good luck!
Carolina
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