Cfi apo tirf 100 oil objective lens

For indirect immunocytochemistry, HeLa cells were grown on 12-mm coverslips and fixed with cold 100% methanol for 10 min. The cells were then permeabilized and blocked with 3% BSA in 0.5% PBST for 15 min. Primary antibodies were diluted in 3% BSA in 0.5% PBST, incubated at room temperature for 1 or 2 h and coverslips were washed three times in 0.1% PBST. Secondary antibodies were diluted in 3% BSA in 0.5% PBST, incubated for 30 min at room temperature and washed again three times in 0.1% PBST. For DNA staining, DAPI solution was used at the final step for 4 min. The coverslips were mounted on slides and observed with a fluorescence microscope (Olympus IX51) equipped with a CCD camera (Qicam fast 1394, Qimaging). Images were processed using ImagePro 5.0 (Media Cybernetics, Inc.) and statistic analyzed with Sigma Plot (Systat Software, Inc.).
To measure the length of centrioles, we used a super-resolution structured illumination microscopy (SIM; Nikon N-SIM) equipped with a CFI Apo TIRF 100× oil objective lens (NA1.49) and iXon DU-897 EMCCD camera. The images were taken as Z-stack with distance between planes of 0.1 μm. The center of the signal was determined by intensity profile in NIS-Elements software. The antibodies were conjugated with Alexa Fluor 555 dyes (Molecular Probes).