Fig. 1: Integrative platform for axotomy and visualization of EpoB-mediated axon regeneration.

a, The experimental pipeline developed in this study. (1) Thalamus tissue was dissected from embryonic day 15.5 (E15.5) embryos and cultured on electron microscopy (EM) grids (top) and glass-bottom dishes (bottom). (2) Explants extended axons onto EM grids (top; scale bar, 200 µm) or glass-bottom dishes (bottom; scale bar, 200 µm), showing axon-dominant neurite extensions (middle; scale bar, 100 µm). (3) Upon maturation, a thin needle was used to cut the targeted axon (scale bar, 50 µm); the overall procedure is shown in Supplementary Video 1. (4) Cryo-EM or cryo-ET (top; scale bar, 200 nm) and light microscopy (bottom; scale bar, 100 µm) analyses were performed. b, Explant growth on electron microscopy grids. Explant images were acquired at different time points using a 10× objective and the longest neurite length from the explant centre was measured. n = 20 (DIV 1), n = 16 (DIV 2), n = 15 (DIV 3), n = 14 (DIV 4), n = 15 (DIV 5), n = 26 (DIV 6), n = 14 (DIV 7) and n = 12 (DIV 8). Data are mean ± s.e.m. c, Axon regeneration in the presence of EpoB recorded by differential interference contrast microscopy, showing the neurite tip at axotomy (Ax, t = 0 min, asterisk). Scale bars, 20 µm. Video of regenerating axons (Ax⁺EpoB⁺) and retracting or stalled axons (Ax⁺EpoB⁻) (control) are available in Supplementary Video 2. d, Quantification of axon reactions after axotomy. n = 29 (Ax⁺EpoB⁺) and n = 33 (Ax⁺EpoB⁻) axons. e, Snapshots of regenerating axons labelled with SiR-tubulin (Ax⁺EpoB⁺ and Ax⁺EpoB⁻). The mild microtubule-stabilizing effect of SiR-tubulin was negligible in the control (Ax⁺EpoB⁻) axons. Scale bar, 5 µm. f, Quantification of axon length over time. Start point is the axotomy site. Data are mean ± s.e.m. n = 29 (Ax⁺EpoB⁺) and n = 33 (Ax⁺EpoB⁻).

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