Polysine microscope adhesion slides

Mice were anesthetized and perfused transcardially with sucrose 5% and paraformaldehyde 4%. Brains were removed and post-fixed overnight in paraformaldehyde 4%. The day after three washes with PBS were performed and brains were incubated for at least eight hours in 30% sucrose. Finally, brains were included in cryomolds with Tissue-Tek OCT compound (Bio-Optica) and put at − 80 °C until cryostat sectioning. Brains were cut with a cryostat and 20 μm-thick coronal slices were collected on polysine microscope adhesion slides (ThermoFisher). Slices were incubated first in blocking solution (3%BSA, 10% goat serum, 0.4% Triton-X-100, diluted in PBS) for at least 30 min and then they were incubated with primary antibodies overnight at 4 °C. Subsequently, three wash (10 min each) with PBS were performed and brain slices were incubated with fluorophore-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After the antibody incubation, brain slices were washed and 4',6diamidino-2-phenylindole (DAPI) staining (ThermoFisher) was carried out (DAPI diluted in PBS to a final concentration of 0.5 μg/ml). Finally, another washing step is performed before mounting the coverslips with Mounting Medium (Vecta Shield). Primary antibodies used were: anti-Parvalbumin (Swant, GP72), Shank3 (homemade SHANK3rb [35 (link)]).

H1299/CDDP cells (5 × 10⁴) were cultured on Polysine microscope adhesion slides (Thermo Fisher) at 37 °C, 5% CO₂ for 24 h. Then, cells on the slides were fixed in 4% paraformaldehyde and blocked with 2% BSA containing 0.1% Triton-X 100. After incubated with primary antibodies against LC3 overnight at 4 °C, cells were further incubated with FITC-conjugated secondary antibody. Afterwards, cells were washed and stained with 4′,6-diamidino-2-phenylindole (DAPI) (Solarbio), imaged with a confocal laser scanning microscopy.