Hcx pl apo cs 63x 1.2 water objective

For microscopic analysis HEK293T cells were mounted on poly-L-lysine coated glass slides. Forty-eight hours after transfection, the cells were fixed with 4% formaldehyde in phosphate buffered saline (PBS) for 10 min. After permeabilisation with 0.5% Triton X-100 for 10 min, the cells were incubated with 3% bovine serum albumin (BSA) in PBS for 30 min. After 1 h incubation with the primary antibody (1/200 diluted mouse monoclonal antibody ANTI-FLAG M2 (Sigma-Aldrich) in PBS supplemented with 0.05% Tween-20 (PBS-T) and 3% BSA), cells were washed three times with PBS-T. Cells were then incubated with 1/600 Alexa Fluor 594 rabbit Anti-Mouse IgG (Invitrogen) in PBS-T with 3% BSA for 1 h. After washing three times with PBS-T, nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and stored in 90% Glycerol in PBS with 0.25% DABCO. Images were acquired and processed using a Leica TCS SP5 confocal microscope equipped with an HCX PL APO CS 63x 1.2 Water objective. Images were processed using Leica AF and ImageJ (14 (link)).

All confocal and spectral data were acquired using the Leica TCSPC 5 Confocal inverted confocal microscope with a HCX PL APO CS 63 x 1.2 water objective paired with an Argon 488 laser. Just prior to imaging, the cells were loaded with CoroNa Green (0.5 μM) and incubated for 3 minutes at room temperature to allow diffusion into the cell. CoroNa Green was excited at 488 nm (2% of maximum laser power) and the emission captured at 516 nm. For all spectral data acquisition, a detection range of 413 nm—728 nm, scan speed of 100 Hz, band width of 9.7 nm, and 32 detection steps was employed to acquire spectral images with a resolution of 256 x 256 at 12 bits.