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:
- Biotium goat anti-mouse conjugated with CF430 Goat Anti-Mouse IgG (H+L), Highly Cross-Adsorbed | Biotium
– ex 430/emit 498
- 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!