We've found that a PSD anti-rabbit and C1qa anti-rabbit give us the best images in conjunction with goat anti rabbit secondaries and a NGS block, but have been unable to stain for these simultaneously. Is there a way to work around this using multiple secondaries or blocks?
Hi Samuel, check out this paper: You stain with your fluorescent Ab, quench the fluorescent stain and restain with a different Ab with the same (or different fluorophore).
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Hi Samuel,
If antibody penetration is not an issue (meaning if you have thin slices), you could also try to conjugate your primaries with different secondaries before the staining (with monovalent secondaries, this is important).
I'd also consider directly labeling your primaries with fluorescent dyes, this is easily accomplished with a variety of commercially available kits!
Lastly, if you mean that your 1st target is PSD95, I'd really just recommend that you find another antibody raised in a different host species. This is a very very common protein to target and I am positive that there's a lot of good ones that'd work for you.. You could request samples from companies like Santa Cruz, sysy, ThermoFisher, etc and see if they work for you!
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