Fig. 10
Immunocytochemistry of U-87MG-AQP4-ECFP cells after treatment with NMOSD patient sera and human complement. U-87MG-AQP4-ECFP cells were treated with 10% NMOSD patient sera (with or without AQP4-IgG) in combination with active or heat-inactivated human complement. Then, the cells were washed with PBS with 10% heat-inactivated fetal calf serum, and immunocytochemistry was performed. (A) Terminal complement complex (TCC) staining. The cellular surface was stained for TCC deposition by applying mouse anti-C5b-C9 neo (aE11) AlexaFluor594 (Novus Biologicals) with 5 µg/mL. (B) NF-κB component p65 staining. Translocation of p65 from the cytosol into the nucleus was visualized by intracellular immunocytochemistry. Cells were fixed, permeabilized, and blocked. Then, 2 µg/mL rabbit anti-human p65 (D14E12) (Cell Signaling) served as the primary antibody, followed by goat anti-rabbit AlexaFluor647 (Invitrogen, Thermo Fisher Scientific). White arrows indicate p65 translocation into the nucleus. Scale bar = 20 μm. AF, Alexa Fluor; DAPI, 4′,6-diamidino-2-phenylindole; TCC, terminal complement complex.
