Cy3 labeled secondary antibody

The MG-63 cells were seeded into five confocal microscopy special dishes at a density of 2 × 10⁴ cells/dish. After the treatments, the MG-63 cells were washed three times with phosphate buffer solution (PBS) and then fixed with 4% paraformaldehyde solution for 30 min and blocked with 50 mg bovine serum albumin (BSA)/mL in PBS for 30 min. Cells were incubated with primary anti-CB2 antibody (1 : 50) overnight at 4°C and then washed three times with PBS, before incubation with Cy3-labeled secondary antibody (1 : 200, Beyotime, China) for 1 h at room temperature. The 4′,6-diamidino-2-phenylindole (DAPI) staining solution (200 μL) was added into each dish for 5 min, and then the dishes were washed three times with PBS. CB2 expression was observed by a laser scanning confocal microscope (FV10i, Olympus, Japan) (Excitation = 550 nm, Emission = 570 nm).

Cells were grown on coverslips in 6-well dishes. Cells were transfected with NEDD4-siRNA for 48 hours and washed with PBS. Next, they were fixed with 2% (w/v) paraformaldehyde and permeabilized with 1% (v/v) Triton X-100. Afterwards, they were blocked with 10% (w/v) normal goat serum in phosphate-buffered saline (PBS) at room temperature for 1 hour. Finally, they were incubated in one of the primary antibodies overnight at 4°C. The following day, cells were washed and incubated for 1 hour with Cy3-labeled secondary antibody (Beyotime, Beijing, China) at room temperature, and then co-stained with DAPI (Sigma-Aldrich, Shanghai, China) to visualize the nuclei. Images were obtained using a fluorescence microscope at a magnification of 200x.

The cerebral cortical neurons were plated at a density of 2 × 10⁵ cells on 0.01% poly L-lysine-coated sterile coverslips. After treatment, the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 at room temperature for 20 min, blocked dropwise with 5% bovine serum albumin (BSA), and incubated at 37 °C for 30 min. The fixed cells were incubated with mouse anti-LC3B (1:300) or rabbit anti-LAMP2 (1:100) overnight at 4 °C. The slides were then washed three times with phosphate-buffered saline (PBS) and incubated at room temperature with Alexa Fluor 488 or a cy3-labeled secondary antibody (Beyotime, Shanghai, China) at a 1:200 dilution based on the source of the primary antibody. Finally, the cells were stained with DAPI, observed, and photographed using a laser confocal microscope (Leica, Wetzlar, Germany). The images for the colocalization analysis (colocalization of LC3 with LAMP2) were calculated using the JaCoP plugin in Image J after the thresholding of individual frames. The colocalization analysis was performed on three independent studies with 50 cells per condition in each experiment.