Hi Beverly.

So your primary Anti-A raised in rabbit will be targeted by your secondary goat anti rabbit. And your Anti B raised in mouse will be targeted by your goat anti mouse. And then you only need to block with NGS.... Perfect. 

However, if you get some nonspecific binding from your Anti-B raised in mouse in your mouse cell, you might want to think about getting a mouse-on-mouse kit or trying a primary from a different host and then choosing another appropriate secondary (I presume you have checked that your mouse antibody reacts with the target mouse antigens on the vendors site?) -

And as for you second question, yes, if you use 2 separate host species, it is safer to use both appropriate blocks - personally, I would run the protocols consecutively in this case - but that's only because I am very cautious and like to trace exactly where a thing may be going wrong and I think its easier to trace sequential protocols than cocktails!  Certainly when optimising! However I work more with sections and don't know how well fixed your cells are to your slides!

I hope this helps and I wasn't misconstruing the question, but if you have anything further - Id be happy to try to help you think!!

I agree with Pia and Victor. Anyway, NGS would be the optimal choice if you use secondary Ab raised in goat, but if you have other serums ( FBS for instance) you can try to do the blocking with those one as well. Just remember to put a negative control for the secondary antibody (where you put only secondary, not primary ab). It often worked fine for me! 

Dear Beverley,

I use commercial diluents instead of sera for blocking as well as for 1' and 2' antibodies dilution. Also, I use both in cocktails, which shortens the procedures quite a lot.

Theoretically,  yes, one should block with a serum from the same species as the antibody. However, I haven't seen much difference in serum block, regardless of the serum/diluent. Here the "if" is, when the primary antibody is "good" enough.

Higher signal-to-noise ratio can be achieved  in antibodies with low specificity by use of the same species serum, however that is the only case, according to my experience.

Best,

Ivan

I have simultaneously stained the 2oAbs for rabbit and mouse 1oAb, with good results.  Just make sure you optimise each individually, and as long as the antigen retrieval conditions and the choice of serum block is the same (BSA or Donkey maybe?) then away you go. 

Good Luck :)

I gues your first hypothesis is correct. You can visualize both antibodies with two different fluorochromes

It is very easy to check: use only one primary antibody (e.g. protein A) and both secundary antibodies in green and red and check in this way for cross-reactivity. If you only see one colour, all is fine. You should also check secondary antibodies only on your sample to find out if you have unspecific binding of these antibodies. Unspecific binding can be controlled with different blocking solutions.

Good luck! Cheers

Tilo

I would stain for each antigen separately to start with, so that you know what to expect. If you use serial sections, it should be possible to match them up and check which cells are labelled. Then when you do the simultaneous staining, it should look similar to the 2 separate results combined.  Everyone has given good advice, I hope it works. 

You could achieve your goal by several ways although having those two antibodies from different host species is the most straightforward way.

There is a good summary in Jackson immuno website: https://www.jacksonimmuno.com/technical/products/protocols/double-labeling-same-species-primary

You can purchase and try fluorophore or HRP conjugated antibodies if available or even try to do it by yourself using commercial kits .

Another possible solution is to use alpaca's secondary VHH antibodies. Please see details here: https://nano-tag.com/protocols/multiplexing