Figure 2. Internalization of AGAb from presynaptic membranes at NMJs occurs rapidly at physiological temperature (37°C).

(A) TS muscle was labeled with anti-GD1b Ab on ice and subsequently incubated at 37°C for 30 and 60 minutes to allow endocytosis. Surface anti-GD1b Ab was detected with secondary Ab and analyzed by fluorescence microscopy. Arrowheads indicate the expected site of surface anti-GD1b Ab signal overlying the acetylcholine receptor (AChR) signal at the NMJ upon incubation at 37°C. (B) Following microscopic analysis, the same TS preparations were permeabilized (with Triton X-100), and the total internalized and surface anti-GD1b Ab was imaged using secondary Ab. (C and D) Fluorescence images for anti-GD1b Ab were quantitated using ImageJ software. (C) Anti-GD1b Ab was rapidly depleted from the cell surface over time. (D) Following permeabilization, no significant difference in total anti-GD1b Ab signal overlying the NMJ was observed, compared with surface Ab levels at t = 0. (E and F) For anti-GD1a Ab (images not shown) rapid clearance by internalization from the cell surface was also observed (E), and by 60 minutes, there was also a mild but significant overall reduction in total anti-GD1a Ab over the NMJ (F, 60-minute time point), suggesting clearance away from this site. At least 150 NMJs were analyzed per time point, and results are from 3 independent experiments per Ab. n = 3; **P < 0.005. Scale bars: 20 μM. Control tissues exposed to secondary Ab alone showed no significant binding or uptake (for anti-TNP IgG3 control data, see Figures 7 and 8).