Hi there, I am trying to do two-channel light sheet microscopy for a whole mouse oviduct, but the autofluorescence from the vessels is masking my true target. I am currently using PFA for fixation and 488 secondary. Does anyone have suggestions for minimizing autofluorescence in whole mount samples?
Hi Avery, I would recommand to use a different fluophore instead of 488 because with the use of this fluorophore the autfluorescence is very strong. You can also try to counterstain your tissue with Sudan Black after you have done your immuno.( destilled water - rinsing; 70 % ETOH - rinsing; 1% Sudan Black solution in 70 % ETOH for 10 min; several rinsings in destilled water; or if the staining ist too dark, short rinse in 70% ETOH before rinsing in water) .
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