Fig. 3.

Cluster formation of AQP4 by binding of E5415A on an astrocytic membrane. Primary cultured astrocytes treated with E5415A (A, C, E, and G) or E5415B (B, D, F, and H) for 10 min (A and B), 1 h (C and D), or 3 h (E and F), or 24 h (G and H) at 37 °C were cooled to 4 °C and further treated with Alexa-Fluor-488-labeled anti-mouse IgG (green) at 4 °C for 1 h. Then cells were fixed with 4% PFA and permeabilized to detect intracellular mAb with Alexa-Fluor-555-labeled anti-mouse IgG (magenta). Bar=5 µm. The lower panels are global images of the upper panels. Magnified areas are indicated by white boxes. Bar=10 µm. (I) Quantification of the number of dots on the cell surface of primary cultured astrocytes incubated with E5415A (red bars) or E5415B (blue bars) followed by visualized with Alexa-Fluor-488-labeled anti-mouse IgG. Fifteen to twenty one images from five cells were taken in each experiment. Numbers of dots in 400 µm² were counted using ImageJ2 (National Institute of Health, Bertesda, MD, http://imagej.nih.gov/ij/) software. Values are means±SD of four independent experiments. **(P<0.01), significant difference versus cells treated with E5415A for 10 min; ## (P<0.01), significant difference between the groups; and NS, not significant as determined by the Tukey-Kramer method.