Fluoview confocal microscope

LNCaP cells were cultured in 8-chamber plates, treated with DRP-27 (10 μM) and incubated at 37 °C and 5% CO₂ for 48 h. Cells were fixed (4% formaldehyde) for 15 min and blocked with 5% goat normal serum (Invitrogen) with 0.3% Triton X- 100 (Sigma–Aldrich) in PBS. Cells were washed (PBS) and incubated with primary antibodies (1:200) (pH2AX and cleaved caspase-3) for 1 h. After three successive washings, cells were treated with either 0.1 μg/ml of anti-mouse IgG or secondary anti-rabbit IgG conjugated with FITC for 1 h. Cells were counter stained with DAPI (30 nm) for 10–20 min, washed with PBS and a coverslip with Fluorogel (Electron Microscopy Sciences, Hatfield, PA, USA) was prepared for visual inspection with an Olympus FluoView confocal microscope. The quantification of the image was done using ImageJ software.

The slides for immunofluorescence were prepared by diluting cells (4 × 105 cells/ml) in PBS. 200 microliters of cells were spotted on microscope slides by cytospin at 1,000 rpm for 10 minutes at room temperature. After cytospin, the slides were transferred into a coplin jar containing ice-cold PBS incubate for 5 min; transferred into ice-cold CSK buffer (10 mM PIPES, pH 6.8; 100 mM NaCL; 300 mM sucrose; 3 mM MgCl2) incubate 1 min; transferred into ice-cold CSKT buffer incubate for 5 min; transferred into ice-cold CSK buffer for 1 min; transferred into 4% paraformaldehyde in PBS incubate for 10min at room temperature. Immunofluorescence was performed by immersing slides in PBST (1x PBS with 0.1% Tween-20) for several minutes. Slides were overlaid with 250 microliters of blocking solution (1x PBS, 1% fetal bovine serum, 0.1% Tween-20) for 1 hour at room temperature. Blocking solution was removed and 100 microliters of primary antibody was added to the cells and incubated for 3 hours at room temperature. The slides were washed 2 times in coplin jars with PBST for 5 min each at room temperature. Secondary antibody was overlaid to spotted cells for 1 hour in the dark. Slides were washed in coplin jars with PBST 2x at room temperature. DAPI was added and the slides were sealed with a coverslip. Imaging was performed using an Olympus Fluoview confocal microscope.

Yeast cells expressing Hxt1-GFP were stained with FM4-64 (lipophilic styryl dye to stain the vacuolar membrane, 1µg/ml) and analyzed with Olympus FluoView confocal microscope under 63× oil immersion objective lens using GFP or Texas Red filter. Images from confocal microscope were captured by FluoView software (Olympus). At least 200 cells showing the respective makers (e.g., FM6-64) were analyzed per each condition. Standard deviations were calculated from three or more independent experiments and are shown as error bars.