Mouse intestinal organoids ; After fixation and storage in PBS-T, when I checked in microscope, the organoid show fluorescent color blue even if I have not added DAPI. Does anyone have such experiences?
Thank you
Effects of fixative, PFA fixed tissues are faintly autofluorescent in the range between 300-500. As long as your DAPI signal is strong you don't need to worry about it the nuclei signals would be much stronger than the background allowing you to take short exposures (low gains) and cutting the background away. If you have strong autofluorescence comparable to DAPI signals that something in these organoids reacts with UV/Blue light. The natural solution using different nuclear staining outside of the Blue spectrum. There is a number of alternative chemicals to stain nuclei outside of the Blue part of spectra such as:
1. YO-PRO Y3603 (491 nm, green), SYTO 17, RedDot™2 Far-Red Nuclear Stain, and even PI. Or you can simply use Histone H3 antibody and then any of the secondaries of your choice outside of Pacific Blue/DAPI channel.
PFA definitely has an effect in producing fluorescence as Anton mentioned. Other sources of autofluorescence for an organoid can be chromatin (if localized in the nucleus) that has autofluorescence in the same region. Besides this, collagen is a strong source of autofluorescence in this zone (UV excitation, blue emission), which can either be produced by the cells (localized within the cell cytoplasm) or globally, from the matrix you used to make your organoid (matrigel, collagen, tissue explants).
In case you want to eliminate PFA based autofluorescence you can simply use a methanol/acetone fixation method. If you want to remove organoid autofluorescence in general you can use reducing agents like sodium borohydride, Sudan black, eriochrome black T.