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Fig. 3 The SPoT reporter reveals translation sites along axons. Representative examples of sites of protein synthesis within axons revealed by HA/Venus immunostaining of Danoprevir-treated embryos. (A–I) Superimposed Venus (green) and HA (magenta) immunostaining signals. (A′–I′) HA immunostaining signal intensity color-coded with imageJ ‘Fire’ lookup table. Color-intensity correspondence is represented in the calibration bar. (A,B,E) Lateral views of the trunk region. (C,H) Lateral views of the tail region. (D) Dorsal view of the anterior head region. (F) Isolated spinal motor neuron; the distal part and growth cone of a large reticulospinal axon is visible, but has no HA staining. (G) Lateral view of isolated RB, motor and inter neurons of the spinal cord. (I) Peripheral arbors of sensory neurons innervating the skin. Note the complete absence of HA signal. Arrowheads mark sites with characteristic accumulation of newly synthesized SPoT reporter in axon initial segment. Arrows point to axons with newly synthesized protein all along the shaft length. Empty arrows point to prominent axons with no protein synthesis. CiA, circumferential ascending neuron; MN, motor neuron; RB, Rohon-Beard neuron; RS, distal portion of a reticulospinal projection. (A,B,D,F,H,I) Transgenic line, Tg(SPoT_chicken ß-actin). Danoprevir treatment time, 1.5 h. (C,E) Transgenic line, Tg(SPoT_tubb5). Danoprevir treatment time, 2 h. (G) Transgenic line, Tg(SPoT_tubb5). Danoprevir treatment time, 40 min. Scale bars: 20 µm. Insets represent twofold magnification of the dashed boxes.