Actin filament distribution in the AIS. (A and B) DIV3 (A) and DIV10 (B) neurons stained with Alexa Fluor 488–phalloidin (F-actin) and AnkG antibody after detergent extraction. Boxes are enlarged on right and shown as individual channels and a merged image. Red and cyan arrowhead pairs mark the AIS and adjacent axonal region, respectively, as examples of how regions of interest were selected for quantification in C. (C) Quantitative analysis of mean phalloidin fluorescence intensity (in arbitrary units) within AnkG-positive AISs of DIV7–21 neurons, relative to distal axon regions immediately adjacent to the AIS. Data were analyzed using two-tailed, paired t test (n = 10 axons per age group). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent standard deviations. Bars: (A and B) 10 µm; (enlargements) 1 µm.