Indirect Immunofluorescence Assay for AQP4 Detection

Cell-based indirect immunofluorescence assay was performed as described previously with modifications (31 (link)). Human embryonic kidney (HEK) 293 cells (ATCC) were seeded onto 8-well chamber slides at 10,000 cells per well and cultured in DMEM in 5% CO₂ at 37°C. They were transfected with pAQP4 using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Cells were fixed with 4% paraformaldehyde, washed with PBS, and blocked with 1% BSA in PBS. Sera of mice were diluted at 1:10 with PBS containing 1% BSA. Commercially available rabbit anti–mouse AQP4 antibody (catalog A5971, Sigma-Aldrich) was diluted at 1:500 and used as positive control. To remove nonspecific antibodies, diluted sera were incubated with rat liver powder at room temperature for 1 hour and then centrifuged at 16,900g for 15 minutes. Next, 100 μL of the diluted sera were added to each well and incubated at 4°C overnight. After washing with PBS, cells were incubated with either Alexa Fluor 488–conjugated anti-mouse IgG (for samples) (catalog A21202) or anti-rabbit IgG (for positive control) (catalog A11070) secondary antibody (all from Thermo Fisher Scientific) at room temperature for 1 hour. Cells were coverslipped with antifade reagent containing DAPI (Thermo Fisher Scientific).