Mouse anti zo 1

Cells in 4 or 8-well chamber slides were fixed in 4% formaldehyde (Ted Pella Inc.) for 10 min, permeabilized in PBS-0.5% Triton for 10 min, blocked for 60 min using 10% goat serum (Invitrogen), and incubated overnight with primary antibodies (in blocking buffer) all at 4 °C. Primary antibodies were rabbit anti-HMGB1 (Abcam), mouse anti-γH2AX (Millipore), rabbit anti-caspase-3 (Cell Signaling), mouse anti-ZO-1, mouse anti-keratin 18 (Santa Cruz Biotechnology), and rabbit anti-vimentin (NeoMarkers). Cells were washed, incubated with secondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor goat anti-mouse 594; Invitrogen) for 30 min at RT, and washed 3× in PBS. The final wash contained 0.1 mg/ml DAPI. We mounted slides in Vectashield (Vector Labs). Images were obtained using an Olympus BX20 fluorescence microscope and Spotfire software (Diagnostics Instruments), and processed with Photoshop CS (Adobe).