Lsm510 meta duo scan laser scanning confocal microscope

For confocal fluorescence analysis, cells after treatment were fixed in 3.7% formaldehyde for 15 minutes at room temperature, blocked in 3% BSA in 0.1% Triton X-100 in PBS. To detect SAHF formation in cultured cells, fixed cells were incubated with rabbit anti-K9M-H3 antibody at 4 °C overnight, followed by anti-rabbit secondary antibody conjugated with Alexa Flour 488 at room temperature for 4 h. Cells with condensed imaging of K9M-H3 that co-localized with DAPI in the nuclei were considered as SAHF positive, and were expressed as percentage of SAHF positive cells versus total cell numbers. For detecting mitochondrial morphology and LC3β expression in NHEKs, cells were first stained with Mito Red at 37 °C for 30 min, and then incubated with rabbit anti-LC3β antibody at 4 °C overnight, followed by secondary anti-rabbit antibody conjugated with Alexa Flour 488 at 37 °C for 1 h. After secondary antibody incubation, A375 or NHEKs cells were stained with DAPI for 10 min at room temperature and washed three times in PBS. Images were acquired by a Zeiss LSM510 Meta Duo Scan laser scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany).

For confocal fluorescence analysis, A549 cells were plated on glass coverslips placed in 6-well plates for 24 h, pretreated with 5 μmol/L shikonin for 2 h, added 200 nmol/L CPT for 0, 1, 4, 8 and 12 h. The cells were fixed in 3.7% formaldehyde for 15 min at room temperature, blocked with 3% BSA in 0.1% Triton X-100/PBS at room temperature for 1 h, washed three times with 0.1% Triton X-100/PBS, and incubated with anti-pRPA antibodies at 4 °C overnight, followed by anti-rabbit secondary antibody conjugated with Alexa Flour® 488 at room temperature for 1 h in dark. After secondary antibody incubation, cells were stained with DAPI for 10 min at room temperature and washed three times with PBS. Images were acquired by a Zeiss LSM510 Meta Duo Scan laser scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany).