Na uplan apo water immersion objective

Cells were seeded in 12-well plates at a density of 1 × 10⁵ cells per well on coverslips overnight. Cells were treated as described above and were fixed in ice-cold methanol and permeabilized for 10 min with 0.5% Triton X-100/PBS, washed with PBS, and then blocked with 5% BSA/PBS. Cells were incubated overnight with primary antibodies diluted in 4% BSA and 0.1% Tween 20 in PBS. Cells were then washed with PBS and incubated with fluorescent secondary antibodies Alexa Fluor 488 goat anti-mouse and Alexa Fluor 555 goat anti-rabbit antibodies were from Molecular Probes (Invitrogen, Waltham, MA, USA). Negative controls were carried out using PBS instead of primary antibodies. Cells were washed, mounted, and imaged by confocal laser scanning microscopy, which was performed with an Olympus Fluo view FV 1000 microscope, using an Olympus 60x/1.20 NA UPLAN APO water immersion objective.

Cells were seeded in 12 well plates at a density of 1 × 10⁵ cells per well on coverslips overnight. After cells were transiently transfected with pcDNA3HO-1 or empty vector, cells were fixed in ice-cold methanol for 20 min at 4 °C and permeabilized with 0.1% Triton X (v/v) in PBS for 5 min at room temperature. After washing twice with PBS, cells were blocked with 3% (m/v) bovine serum albumin (BSA)/PBS for 1 h at room temperature. Cells were incubated overnight at 4 °C with anti-MX1 and HO-1 antibodies diluted 1:200 in 3% (m/v) BSA/PBS. Negative controls were carried out using PBS instead of primary antibodies. Cells were washed with PBS and incubated with fluorescent secondary antibodies for 1 h at room temperature, then stained with DAPI, and imaged by confocal laser scanning microscopy, performed with an Olympus Fluo View FV 1000 microscope, using an Olympus 60X/1.20 NA UPLAN APO water immersion objective.