Extended Data Fig. 1: Pearled morphology is observed for all axons except after chemical fixation.
From: Membrane mechanics dictate axonal pearls-on-a-string morphology and function
a. Example micrographs showing acutely extracted mouse brain tissue either high-pressure frozen (left) or chemically fixed (right). The boxed axons are reconstructed using IMOD. Note that chemically fixed axons are like cylindrical tubes. Scale bar: 500 nm. b. Higher magnification images of the boxed area in a. Scale bar: 200 nm. c. AiryScan images of axons live without (left) or after 5 min of fixation (right). Inset shows synapses marked by RIM1a-CFP. Arrowheads mark synapses, arrows mark NSVs, circle marks extreme axon pearling associated with degenerating axons. Scale bar: 5 µm. d. Further images of an axon from a separate culture live without (middle) or after 5 min of fixation (bottom). Synapses marked by RIM1a-CFP (top). Arrowheads mark synapses, arrows mark NSVs. Scale bar: 1 µm. e. Additional representative images of axons from acutely extracted brain tissue, organotypic slice culture, and dissociated neuron culture. Scale bar: 200 nm. f. Super Plots of Fig. 1d, showing experimental variability. Median and 95% confidence intervals are shown. N = 3 and n = 30 axons from each acutely extracted sample, n = 133 axons from the organotypic sample, N = 3 and n = 100 axons from each dissociated sample. Kruskal-Wallis test, followed by Dunn’s multiple comparison test was used. Each color represents one replicate. Each dot is one axon. g. Frequency distributions for acute, organotypic, and dissociated culture of either NSV lengths or connector lengths. h. Additional STED micrographs showing pearled axon morphology in live neurons from organotypic slice cultures. Scale bar: 500 nm. Individual axons are straightened and represented on the right panels. Scale bar: 200 nm.
