Never encountered this problem before (been a tech for 5 years), but here goes:
Brain tissue derived from a perfused rat, stored in 4% PFA for roughly 4 days, then moved over to 30% sucrose in 1x PBS for 4 days, then sliced on a cryostat at 40um.
Averaging 9 pieces of free-floating tissue per well (regions are OFC-PFC, so small - typically do about 6 slices of free floating tissues in further back regions like dSTR or HPC).
Tissue was then washed 3x for 10min in PBS (hereafter referred to PBST), transferred to 0.5% donkey serum in 0.1% Triton x100 in 1x PBS. Tissue is blocked for three hours, antibodies are added and washed at 4C for 48 hours. Following, tissue is washed 3x in PBST, and blocked in 0.5% donkey serum in PBST with secondary for two hours. Tissue is washed three times in PBST for 10min each round, followed by a final 10min wash in PBS.
All of my tissue is stuck together or to itself, sticks to my paint brushes (both synthetic and natural bristles) and refuses to unfold.
I've tried washing it longer in PBST and PBS and even took a slice and vortexed it in PBS for giggles. My solutions are all pH 7.2-7.4. I am at a loss.
Any suggestions as to why this is happening, or better yet, how to unstick the tissue?
Many thanks for your thoughts!
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