Cfi plan apochromat vc objective

NIH/3T3 cells (American Type Culture Collection, ATCC CRL-1658) were cultured under standard conditions in DMEM supplemented with 10% fetal bovine serum (FBS). Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min. After washing in PBST (PBS with 0.1% Tween-20), cells were subjected to permeabilization with PBS containing 0.2% Triton X-100 for 10 min and then blocking with 3% BSA in PBS for 40 min. Primary antibody, anti-human FAM50A antibody (1:200; Novus Biologicals, NBP1-89344), which has cross-reactivity to mouse Fam50a protein^{54 (link)}, was treated in PBS containing 0.1% Triton X-100 at 4 °C overnight. Cells were then washed in PBST and incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:1000; Invitrogen, A-11008) and Hoechst 33342 (1:10,000; Invitrogen, H3570) at room temperature for 1 h. After washing with PBST, the cells were subjected to confocal imaging by using a Nikon A1R confocal microscope (Nikon Instruments) equipped with a Nikon CFI Plan Apochromat VC objective (×60/1.4 numerical aperture (NA); Nikon Instruments) and digital zooming of Nikon imaging software (NIS-element AR 64-bit version 3.21; Laboratory Imaging).

Live-cell imaging was performed using a Nikon A1R confocal microscope (Nikon Instruments) mounted onto a Nikon Eclipse Ti body equipped with a Nikon CFI Plan Apochromat VC objective (60×/1.4 numerical aperture [NA]; Nikon Instruments) and digital-zooming Nikon imaging software (NIS-element AR 64-bit version 3.21; Laboratory Imaging). A Chamlide TC system placed on a microscope stage was used for maintaining environmental conditions at 37 °C and 10% CO₂ (Live Cell Instruments). Photoexcitation was delivered using a photostimulation module in Nikon imaging software (NIS-elements) that provided one loop of 1 s stimulation with a 488-nm laser. A light power density of 490 μW mm⁻² (measured with an optical power meter from ADCMT) was used for photoexcitation, unless stated otherwise.