Lsm700 upright microscope

Time-lapse imaging was performed using a Carl Zeiss LSM780 inverted microscope with a Plan-Apochromat 20×/0.8 at 37°C under 5% CO₂. Imaging of fixed samples was performed using a Carl Zeiss LSM700 upright microscope with a Plan-Apochromat 20×/0.8. Live imaging was recorded with a frame rate of 10 min.

After sequence verification, ACR and KCR construct variants were cloned into the multiple cloning sites of a pcDNA3.4 vector (Genscript) by XhoI and EcoRV restriction enzyme digest. Then, 250 ng of the respective DNA constructs were transfected into N2a cells⁵⁴ using Lipofectamine 3000 (Invitrogen). The cells were left to incubate in serum-free media for 48 h. The N2a cultures were then washed three times with PBS and fixed for 20 min at room temperature with 4% paraformaldehyde diluted in PBS-Triton X-100 (0.25%, 85111 Thermo Fisher Scientific). After fixation, the cells were blocked in 5% BSA (A-420-500, Gold Biotechnology) diluted in PBS-Triton X-100 (0.25%) for 1 h at room temperature and stained for GFP (Abcam ab13970, RRID: AB_300798) at 1:2000 v/v dilution for 1.5 h at 37°C. Afterward, the cultures were rinsed three times with PBS and incubated with an Alexa 488 goat anti-chicken (A-11039 Thermo Fisher Scientific, RRID: AB_2534096) at 1:500 v/v dilution for 1 h at 37^oC. Finally, the cells were washed three times with PBS and mounted onto microscope slides in Vectashield Vibrance mounting media (H-1700 Vector Laboratories, Burlingame, CA). Imaging was performed on a Zeiss LSM700 upright microscope using a 100× objective. Maximum intensity projections were obtained after image analysis with ImageJ.