Goat anti aqp4 antibody

The ELISA was performed as previously described by Pisani et al., 35. Briefly, Maxisorp NUNC Plates (Thermo) were coated with 0.2 μg of commercial goat anti‐AQP4 antibody (Santa Cruz Biotechnology, sc‐9888) overnight at 4°C or for 2 hrs at 37°C. After coating, wells were washed and coated with approximately 35 ng of AQP4. Negative control wells were only coated with the buffer. After incubation for 1 hr, sera diluted from 1:1000 to 1:8000 and goat anti‐AQP4 were incubated in the wells for 1 hr under shaking. Wells were then washed, incubated with anti‐human biotinylated secondary antibody (Millipore AP112B), washed again and incubated with streptavidin‐HRP (Millipore SA202, 1:1000 in A). After 1 hr, 100 μl of TMB solution was added (Millipore) for 20 min., and the reaction was stopped by adding 100 μl of 0.3M sulphuric acid solution. Finally, absorbance was read at 450 nm. Normalized absorbance was calculated as follows: absorbance of AQP4‐coated well minus absorbance of negative control well. Absorbance was read using a Flex Station 3 (Molecular Devices, Sunnyvale, California, USA).