Cell culture, transfection, and observation of cells expressing EGFP-MAPs

SH-SY5Y cells were maintained in Dulbecco’s modified Eagle’s medium (Wako Pure Chemical Industries) supplemented with 10% fetal calf serum and 0.001% penicillin/streptomycin at 37 °C in 5% CO₂. The cells were cultured on a round coverslip (Matsunami Glass, Osaka, Japan) coated with poly-d-lysine (0.1 mg/mL) and transfected with pEGFP-MAP4, pEGFP-MAP2, or pEGFP-tau, which are mammalian expression plasmids encoding full-length MAP proteins with its C-terminal fused to EGFP. In this transfection, we used SuperFect transfection reagent (QIAGEN) according to the manufacturer’s instructions. The cells were cultured for 1 day, after which the coverslips were treated with 4% paraformaldehyde phosphate buffer solution (Wako Pure Chemical Industries) for 20 min at 20 °C and washed twice in PBS. Cells were then treated with PBS containing 0.2% Triton X-100 for 5 min and washed three times in PBS. When acetone fixation was used, cells were treated with acetone at − 20 °C for 15 min and not permeated with Triton X-100. After blocking with PBS containing 3% BSA (PBSB) for 30 min at 20 °C, the coverslips were incubated in PBSB containing a monoclonal anti-β-tubulin antibody (T4026, Sigma-Aldrich) at a dilution of 1:200 for 1 h and washed three times with PBS for 5 min. The coverslips were incubated in PBSB containing a secondary antibody conjugated with Alexa Fluor 546 (A-11003, Thermo Fisher Scientific) at a dilution of 1:100 for 1 h. Simultaneously, for fluorescent staining of F-actin, the coverslips were incubated in PBSB containing 0.1 mM Alexa Fluor 647 phalloidin for 1 h, washed three times with PBS for 5 min and rinsed with water. The coverslips were mounted using SlowFade Diamond Antifade mountant (Thermo Fisher Scientific). The cells were observed under a fluorescence microscope equipped with a color CMOS camera (DS-Ri2, Nikon) and confocal microscope system (Nikon Eclipse Ti-C2, Nikon). EGFP intensity (mean of gray values) was measured using ImageJ software to compare the expression levels of EGFP-MAPs.