Experience with 440nm excitable fluorophores? Brilliant violet 480, Biotium Cf430, not working

I have a very strange problem. We have a confocal microscope with a laser launch partly optimized for live cell imaging with CFP, so we have a 440nm laser instead of the usual 405nm. We know this laser works because my labmates use it to image CFP all of the time in live and fixed cells.

I am interested in doing immunofluorescence in this channel in FFPE liver. To that end, I have taken an optimized, mouse 1’ antibody that is easily visualized with goat anti-mouse alexa 488 and alexa 568, and tried now two different secondaries which would seem to a perfect match for our 440 laser + CFP filter set:

  1. Biotium goat anti-mouse conjugated with CF430 Goat Anti-Mouse IgG (H+L), Highly Cross-Adsorbed | Biotium
    – ex 430/emit 498
  2. Brilliant Violet 480 goat anti-mouse Secondary Antibodies - Jackson ImmunoResearch
    – ex436/em478 spectrum at bottom of link above

Both are basically perfect excitation/emission matched for CFP. I then performed the usual protocol that works for other secondaries, and saw literally zero signal on both (no primary + secondary appears the same, as does no primary/no secondary). I feel I am missing something really silly here. On the other hand, perhaps there is a reason no one images with these fluors? Both companies have gotten back to me saying these lots passed QC.

I have atto 425 coming soon, but something tells me it’s not the fluor…

I can post pictures, other experimental details if important, thanks for considering!

I don’t know what your setup is, but since you have found the 440nm laser to work in other cases, there is no chance your main beam splitter is currently not set to pass that wavelength on the track you are using? I have occasionally switched lasers on a given track and forgotten to swap the MBS as well, resulting in blank images.

Also, you might try adding a drop of fluorophore solution onto a coverslip and seeing if you get any general emission glow. The 405 later is not optimal but it should also give you some excitation, so you should be able to check with that as well. That means you should be able to double check that fluorophore on another standard system.

This is good advice. I’m working with the guru for our confocal, he is sure it’s not a silly problem but we’re double checking this week.

Really helpful point about trying on a different microscope with a 405, we should be able to try that as well. Thank you for your feedback!

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