Hi Ashish,

are your two mouse antibodies monoclonal antibodies? And if so, did you check what isotypes they are (IgG1, IgG2a, IgG2b, IgG3)? There are very good isotype-specific secondary antibodies (e.g. as part of the Alexa Fluor series at Life), which might still allow you to differentially detect your two mouse antibodies. This should also be the 'cleanest' solution if it is applicable here.

Best,

Dennis

Hi Ashish,

I suppose IFA is immunofluorecence analysis...

I would suggest you to couple your antibodies directly with different fluorophores. for example, you can find there kits and suggestions:

http://www.lifetechnologies.com/de/de/home/life-science/cell-analysis/labeling-chemistry/protein-and-antibody-chemical-labeling/antibody-protein-labeling-kits.html

And for sure, there are different suppliers on the marcet, so maybe search for more, for your convienience.

Best GAD

If you really can't obtain antibodies for your targets raised in different animals, you can try one of the following:

Complex approach...

Incubate with one of your primary antibodies then use a secondary antibody labelled with one fluorophore. You then need to block with an excess of unlabelled secondary, followed by multiple washes to remove unbound antibody. You can then proceed to incubate with the second primary, followed by the secondary antibody labelled with a different fluorophore. When following this approach, it's important to include a number of controls to ensure that any co-localisation is genuine and not a consequence of cross reaction between your primary/secondary antibodies, e.g. omit the second primary to ensure that there's no labelling of the first with the second secondary. There are others, so it quickly gets complicated!

Indirect, but simpler...

Find a third antibody that stains your organelle of interest and was raised in a different animal; carry out double labeling with this and each of the other antibodies. Your third antibody will have a consistent localisation between the two IFAs, so you'll be able to assess the relative localisations of your proteins of interest. I've taken this approach on several occasions and it's a lot simpler than the first!

Good luck.

Hi Ashish,

are your two mouse antibodies monoclonal antibodies? And if so, did you check what isotypes they are (IgG1, IgG2a, IgG2b, IgG3)? There are very good isotype-specific secondary antibodies (e.g. as part of the Alexa Fluor series at Life), which might still allow you to differentially detect your two mouse antibodies. This should also be the 'cleanest' solution if it is applicable here.

Best,

Dennis

I faced a similar problem - and to get rid of it, I was using Isotype-specific Alexa Fluor secondary antibodies from Life Technologies, which was indeed working good.

Dennis suggestion is the best I can find. It happened to me that the two I was using where different Isotype. Jackson labs have very nice specific conjugated antibodies, crossabsorved; expensive but useful.

The best thing to do is find a different species primary if you can or directly label each primary, if not there is one techniqe that I have tried with mixed results; label with the primary and secondary for antibody A and then refix your sample in PFA or Glutaraldehyde and then label with antibody B primary and secondary, that way you are fixing the secondary onto the primary preventing the second (antibody b) secondary from binding, if you see what I mean.

Hi,

Many years ago I had to deal with the same situation. If you know where the expression is and its relative intensity you may use some tricks. The first thing to consider is how important it is to perform the assay with both antibodies at the same time (of course I can guess why).

1-Can't you use two different but identical specimens, one for each antibody? You may overlap the results with photoshop, nobody will crucify you if you say how you've performed the assay and why.

2-If you really have to do it at the same time consider the following:

a) if you start by developing the stronger antibody once you might end up with unwanted background and you must use a more aggressive washing process before developing the antibody B

b) you should develop the staining for the faster antibody to stain your sample.

3-Can't you perform a double RNA in situ hybridization instead?

Just a word from someone who's been around for a while...

Do you really have a problem or are you creating one? Why don't you just do two independent experiments?

Good luck !

See Jacksons immunoresearch technical data, (http://www.jacksonimmuno.com/technical/fab-blok.asp) they have a lot of advise on this. You can digest one your antibodies into Fab fragments or use Fab secondaries

to turn one of your primaries into a different species.

You have answered your question. May be you need the extra blocking step.

Adding on to what Beverley has said, there is an additional option. If no conjugates for either antibody are available (even a biotin conjugate would be very useful), then you can purchase a conjugation kit and conjugate it yourself. Kits range from extremely easy (though somewhat less pure) [Lightning Link;http://www.innovabiosciences.com/] to more complex but quite pure [SoluLink: http://www.solulink.com/solulink-technology].

Vector Laboratories have published a good pdf http://www.vectorlabs.com/data/brochures/MLB.pdf which describes how to use more than one mouse monoclonal antibody in IFA. In the past I have also directly biotinylated one of the monoclonal antibodies and then used an anti-biotin as a second step followed by a fluorescently labelled anti-avidin (from DAKO) as a third step with clean results. Hope this is of interest.

Hi Ashish,

I agree with all the people that suggest to directly conjugate one of the two antibodies you have. Or (and for me is the better way) to use two antibodies from different  species.

Lorena