Hi everybody,
We are interested in dyes that perform well with 2P imaging. We have large structures stained with primary Ab and secondary Ab conjugated to fluorophores. Alexa488 works fine for us, but we would like to extend the palette of colours / multiplex. Has someone experience with this?
Thanks and best regards,
Laurent.
Alexa 647. It has a bit of a unique excitation cross-section; if memory serves, you excite it with 800nm or thereabouts. Good luck!
Alexa 647 is not especially good with 2p: it’s unusually dim and bleaches very fast. But, yes, you can hit it around 800 nm. For far-red with 2p, SeTau-647 is better. That said, I’ve been using these dyes either bound with CTB or with whole-brain labelling then imaged 2p, so this is dimmer than it would be if one did IHC on a slice then imaged.
AF488, 568, and 594 are all pretty good (Two-photon cross-sections). AF555 is bad 2p, don’t use that one.