Methods and Techniques Discussion > Immunofluorescence (IF)

DAPI Smeared in Acetone Fixed Liver Cryosections

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JThomas:
Hello all,

I am staining 6M 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 (6M) and mounted on SuperFrost slides. Air dry for ~2 hours and store at -20C (all slides are stored for less than 2 weeks before staining). Remove slides from -20C and air dry for ~10-15 minutes. Place in -20C 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 -20C? I have heard that any residual water in the section will cause nuclei to burst.

Thanks so much :)
James

AKatharina:
this happens when slides are moved to a wash step immediately after aceton fixation. wait for approx. 60 seconds before proceeding-> solved the problem for me when working with murine spleen!

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