I am attempting to visualise in the same cell sample two different antigenes using two primary antibodies of different species (rabbit monoclonal and a mouse polyclonal). I am wondering if the simultaneous protocol is applicable even if one of the antibodies is polyclonal?
I could find several protocols for sequential and simultaneous staining but I could not find any information if both protocols are applicable, and give the same result?
I have always done simultaneous staining and never had an issue. Since you are doing immunofluorescence I would recommend simultaneous staining as you won't have to worry about losing signal etc and as long as you use two different fluorophores you should be able to visualize them simultaneously.
I agree with Anna. I've only had to do sequential staining when both primary antibodies were raised in the same species.
You have antibodies raised in two different species, so after optimisation of their dilutions (separately) - there is no point in doing sequential staining. Simultaneous staining allows for shorter experiment time, less possibility of cell/tissue detachment and less possibility of accidental exposure to light. I have done parallel experiments with simultaneous and sequential antibodies and simultaneous was better. Good luck with your project.
I confirm that simultaneous stainning is better than sequential if you have the primary antibodies raised in different species. This will anable you to shorten the possible exposure time to light and thus decrease bleaching. It does not matter if one antibody is monoclonal and the other is polyclonal. Have a nice experiment and satisfaying results.
If your primary antibodies are raised in different species, then simultaneous staining can be pursued without any issue. However, if you have to use antibodies raised in same host, then you will have to do a sequential staining. I have personally not had any issues in doing sequential staining vs simultaneous staining. Best wishes.
In your case, I agree with the group that the simultaneous staining will work. also it will save your lot of time. But if one of your primary antibodies is raised in the same host as one of your secondary antibodies (e.g. both were raised from goat), then you have to do the secondary antibody staining sequentially.
One other note, I have found that mouse, rabbit and goat tend to work best as primary antibodies, so I used donkey secondaries, since you will almost never run into a donkey primary. This way, you only have to invest in one set of secondaries, and normal serum.
Although simultaneous staining would be more sensitive, sequential staining would have greater specificity in this context (to prevent hybridisation of the mouse polyclonal to the rabbit monoclonal which might result in a false positive for the antigen supposedly recognized by the mouse pAb). Preference for a particular method is solely dependent on your experimental design & what you intend to do - if quantification of fluorescence intensity, calculation of dynamics, etc were a follow-up procedure, simultaneous staining would not be a good choice in this respect.