Fig. 4

In situ visualization of axonal protein synthesis upon ischemic stress. A, B Metabolic labeling of puromycin in the axonal terminal chamber (A) and Puro-PLA assay (B) monitoring newly synthesized proteins in situ. After axon-restricted puromycin-labeling, a protein-of-interest (POI) is recognized by antibodies against puromycin (red Y) and the POI (black Y). PLA signals (red dots) are detected when PLA-plus and PLA-minus secondary antibodies (green Y and blue Y, respectively) are close enough to be ligated together and amplified by rolling-circle replication. C, D, Representative images (C) and quantifications (D) showing axonal protein synthesis in response to ischemic stress. Cortical neurons at DIV14 were subjected to OGD for 30 min, followed by reperfusion (R) for 0, 4, or 8 hours as indicated. Axons were incubated with 3 μM puromycin (Puro) for 15 min before fixation. The PAK5-Puro-PLA or Miro-1-Puro-PLA signals (red dots) along βIII-tubulin-labeled axons (green) were normalized to total βIII-tubulin signal area. Data were quantified from n = 30–47 images (D) from more than 3 chambers per condition in three independent experiments. Data were expressed as mean ± SEM and analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Scale bars, 10 μm