Comparison of fixation methods of heated U2OS cells.

(A) U2OS cells were heated using the PBS immersion method in pre-heated PBS for 3 minutes at the indicated temperature, then cooled to room temperature for 5 minutes and then fixed with either paraformaldehyde (PFA), PFA then MeOH or MeOH. Nuclei were stained with Hoechst 33342 and quantified by immunofluorescent imaging. Values are the mean of 12 wells ± SD. (B) Chk1 localization was determined by immunofluorescent staining and high content imaging. (C) The mean nuclear Chk1 intensity was quantified from the images obtained in B. Values are the mean of 2 wells.