Extended Data Fig. 3: Quality control of cryo-EM images showing membrane rupture under high tension.

a) Cryo-EM montage of a thalamus axon incubated in hypotonic media conditions before vitrification. The top panel shows a high-magnification montage of an axon. Under hypotonic conditions, membrane tension increases and compromises membrane integrity during the vitrification process. Scale bar: 400 nm. Dotted white lines indicate the positions of zoomed-in insets shown below the montage. A light orange shade highlights the trace of the axon. Scale bar: 200 nm. b) High-magnification cryo-EM montage of a control axon (neurons grown in normal culture media). Scale bar: 400 nm. Zoomed-in insets at different locations were shown below the montage. A light orange shade is used to highlight the trace of the axon. Scale bar: 200 nm. c) Live imaging of axons after axotomy and treated with EpoB. Fluorescently-labelled tubulin antibody (cyan) was added. No microtubule staining, showing the plasma membrane was sealed immediately after axotomy. Scale bar: 20 µm.