I am staining 6µM liver cryosections with Phalloidin AF-488 and using VectaShield with DAPI to label DNA. When I image my slides, the DAPI signal is smeared all over the section as if the nuclei have ruptured and the DNA is spreading across the section. The Phalloidin AF-488 signal, on the other hand, looks beautiful and I can see nice outlines of my cells. Here is my procedure:
OCT embedded liver is cut (6µM) and mounted on SuperFrost slides. Air dry for ~2 hours and store at -20°C (all slides are stored for less than 2 weeks before staining). Remove slides from -20°C and air dry for ~10-15 minutes. Place in -20°C acetone for 10 minutes. 3 X 5 min TBS wash. Apply Phalloidin AF-488 1:200 dilution in TBS containing 1% BSA for 1 hr at RT. 3 X 5 min TBS wash. Apply small drop of VectaShield, coverslip, and image.
Has anyone run into this problem? My only thoughts are to try 10% NBF for fixation (but I am worried about using this when I do dual-staining using Phalloidin and antibody) or to air dry for longer after removing from the -20°C? I have heard that any residual water in the section will cause nuclei to burst.
Thanks so much