In our initial small molecule screen, dechorionated olig2:egfp larvae were treated with 10 μm kinase inhibitor in 1% DMSO in PTU egg water from 24 to 76 h post-fertilization (hpf). Kinase inhibitors used were 1 of 430 kinase inhibitors from the L1200 Kinase Inhibitor Library (Selleck Chem), MK-2461 (Selleck Chem), or Trichostatin-A (TSA) (Selleck Chem). Control siblings were treated with 1% DMSO in PTU egg water. The small molecule screen was conducted in triplicate.
For the EdU incorporation assay, larvae were treated with 0.4 mM EdU in 4% DMSO from 70 to 74 hpf in PTU egg water at 28.5 °C then fixed for 1 h in 4% PFA at RT. Larvae were washed for 5 min with 1X PBSTX, 5 min in DWTX, then permeabilized with cold acetone for 10 min at −20°C and stained for EdU using the Click-it EdU Cell Proliferation kit for Imaging with Alexa Fluor 647 dye (ThermoFisher), as detailed in the kit protocol. Click-it reaction was performed for 1 h at RT and thoroughly washed overnight with PBSTX prior to imaging.
For the EdU incorporation assay, larvae were treated with 0.4 mM EdU in 4% DMSO from 70 to 74 hpf in PTU egg water at 28.5 °C then fixed for 1 h in 4% PFA at RT. Larvae were washed for 5 min with 1X PBSTX, 5 min in DWTX, then permeabilized with cold acetone for 10 min at −20°C and stained for EdU using the Click-it EdU Cell Proliferation kit for Imaging with Alexa Fluor 647 dye (ThermoFisher), as detailed in the kit protocol. Click-it reaction was performed for 1 h at RT and thoroughly washed overnight with PBSTX prior to imaging.