If I have 2 primary antibodies (one raised in mouse, other in rabbit) that I have found to work well individually on whole mounts, would it be expected that these 2 would work as effectively in a sequential double-immunostaining protocol on the same sample? What problems should I anticipate running into, if any?
Thanks
You didn't say if the antibodies are targeting the same or different proteins, and if the epitopes they recognize are at all similar. Assuming no cross-reaction with similar sites or competition for the same site, I anticipate no problems for you.
Try them first sequentially. If this works, to save time you can then try simultaneously (block, add two primaries, rinse, add two secondaries).
Good luck with your experiment.
Sorry, the primaries are targeting different proteins, epitopes are unknown since they were produced by immunising mice with cell populations
I regularly stain tissue sections with different antibodies for two different proteins in a 2 step manner. I sometimes see a diminished signal for whichever antibody I used first which could be due to the subsequent washes required to undergo a second primary/secondary staining step. For me, it's not a huge issue and can be overcome by increasing the concentration of antibodies. Try alternating the order in which you use the antibodies and compare for yourself to see.
Good luck!
Alex
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