Organization of AIS coat after LatB treatment. (A and B) Fluorescence microscopy of DIV10 neurons stained with an AnkG antibody and Alexa Fluor 488–phalloidin (F-actin). Boxed regions are enlarged in bottom panels as individual channels. (A) Control DMSO-treated neuron. (B) Neuron treated with LatB (4 µM for 1 h). (C) Mean phalloidin fluorescence intensity in the AIS of DIV10 neurons after LatB or DMSO treatment. Data analyzed using Welch’s t test (n = 10 axons per treatment group). Error bars represent standard deviations. (D and E) PREM of the AIS in LatB-treated DIV10 neurons after labeling with phalloidin immunogold. (D) The AIS coat at the axon shaft remains apparently intact and contains only short actin filaments (small gold clusters). (E) The AIS coat at the branch junction is sparse and disorganized, with large gaps. Inset shows an overview of this branch junction with axon area shaded in red and the zoomed region marked by green box. Bars: (A and B) 10 µm; (A and B, enlargements) 5 µm; (D and E) 200 nm; (E, inset) 1 µm.