I have a lot of questions about murine skin tissue immunofluorescence (IF).
1. What causes autofluorescence in skin tissue? Even though I don’t use PFA to fix before embedding Into OCT, autofluorescence still exist. So I wonder what on earth causes autofluorescence?
2. Does PFA fixation influence frozen section immunofluorescence by crossing proteins? If so, is antigen retrieval feasible in frozen section IF stain?
Hi Jifan,
Every tissue has autofluorescence, although levels differ per tissue. Most autofluorescence is in the 480-580 nm region. Elastin is well known to be autofluorescent (detectable with settings for FITC).
If you want to perform a single staining, you can best use a fluorophore like Cy5/Alexa647. Autofluorescence is less a factor there.
PFA fixation is possible, but it may block some epitopes.
Mild fixation with 0.5% PFA is a good option, but no guarantee for good results. Antigen Retrieval induced by heat is not an option. Maybe enzymatic retrieval.
My advice:
1.Try a fluorophore with relatively high emission spectrum.
2. If option 1 does not work, try different fixation methods and see what works best.(with or without antigen retrieval)
regards,
Ronald
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