Inverted microscope eclipse ti u b

Cells were seeded onto coverslip in a 24-well plate. After indicated treatment, cells were fixed with cold methanol at −20 °C for 5 min and blocked in 3% bovine serum albumin (BSA) at room temperature for 30 min. Cells were incubated with rabbit anti-GFP antibody (1:500, MBL #598), mouse anti-acetylated tubulin (1:2000, Sigma #T7451) or rat anti-α-tubulin (1:2000, Millipore #MAB1864) at 4 °C overnight, washed with PBS followed by incubation with goat AlexaFluor 488-conjugated anti-rabbit (1:500, Invitrogen #A32731), 568-conjugated anti-mouse (1:1000, Invitrogen #A11031) or 647-conjugated anti-rat IgG (1:000, Invitrogen #A21236) for 2 h at room temperature. After washing with PBS containing DAPI, coverslips were washed with deionized water and mounted with FluorSave (EMD Millipore). Images were acquired by fluorescence microscope (Nikon Inverted Microscope Eclipse Ti-U/B, NY, USA). Fluorescence intensity was quantified by ImageJ software (NIH) as described^{12 (link)}.

After treatment with paclitaxel for 48 h, cells were incubated with H33342 (2 μg/mL) at 37 °C for 15 min in the dark. Cells were imaged at least five random field/groups under a fluorescent microscope (Nikon Inverted Microscope Eclipse Ti-U/B, NY, USA). A number of apoptotic cells to total cell number was calculated and presented as percentage of apoptotic cells.