Sp5 dmi 6000b confocal microscope

In this study, cells were cultured with glass coverslips (Thermo Fisher Scientific; Waltham, MA, USA). Cells were fixed with 3% paraformaldehyde (PFA) in PBS for 15 min, and treated with 200 mM glycine solution to remove the PFA. Subsequently, cells were incubated with 0.2% triton X-100 permeabilization for 15 min and then blocked with PBS–1% BSA with 0.1% sodium azide for 1 h at room temperature, or overnight at 4 °C, to block unspecific binding of the antibodies. Cells were consecutively incubated with two primary antibodies for concurrently protein detection. The first primary antibody was incubated between 5 and 6 h at room temperature and next the second primary antibody overnight at 4 °C. Cells were washed in PBS three times for 5 min. Next, cells were incubated with the secondary antibodies at 1:1000 dilution for 1 h at room temperature in the dark. Cells were washed in PBS three times for 5 min in the dark. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) at 1:1000 dilution for 5 min. Finally, cells were washed three times for 5 min in PBS and slides were mounted with Mowiol. Fluorescent images were acquired with a LEICA SP5 DMI-6000B confocal microscope (Leica; Wetzlar, Germany), using the following lasers: Argon (488 nm), DPSS (561 nm) and UV Diode (405 nm). Images were analyzed using the ImageJ software.

In this study, cells were cultured with glass coverslips (Thermo Fisher Scientific; Waltham, MA, USA). Cells were fixed with 3% paraformaldehyde (PFA) in PBS for 15 min, and treated with 200 mM glycine solution to remove the PFA. Subsequently, cells were incubated with 0.2% triton X-100 permeabilization for 15 min and then blocked with PBS–1% BSA with 0.1% sodium azide for 1 h at room temperature, or overnight at 4 °C, to block unspecific binding of the antibodies. Cells were consecutively incubated with two primary antibodies for concurrently protein detection. The first primary antibody was incubated between 5 and 6 h at room temperature and next the second primary antibody overnight at 4 °C. Cells were washed in PBS three times for 5 min. Next, cells were incubated with the secondary antibodies at 1:1000 dilution for 1 h at room temperature in the dark. Cells were washed in PBS three times for 5 min in the dark. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) at 1:1000 dilution for 5 min. Finally, cells were washed three times for 5 min in PBS and slides were mounted with Mowiol. Fluorescent images were acquired with a LEICA SP5 DMI-6000B confocal microscope (Leica; Wetzlar, Germany), using the following lasers: Argon (488 nm), DPSS (561 nm) and UV Diode (405 nm). Images were analyzed using the ImageJ software.