最近的心结（1）-药物筛选

 The most common or “standard” zebrafish small molecule screening design uses a single concentration (10 µM) in a wild type genetic background, but how this design might be improved by varying and adding to these basic conditions has yet to be systematically explored.

To minimize this possibility we added the compounds after axis specification and gastrulation were completed, just before the formation of the first segment. 

 We therefore tested each small molecule at three different concentrations (2, 10, and 50 µM) from 10 hpf to 36 hpf, which covers the developmental interval of segmentation without interfering with early embryonic development.

Small molecule treatment and technical handling

Embryos of the appropriate genotype were distributed into MultiScreen-Mesh 96-well plates (Merck Millipore) before 10 h post fertilization (hpf) and incubated in E3 until the small molecule add-in. 243 small molecules were selected from libraries provided by Calbiochem (StemSelect® Small Molecule Regulators 384-well library I, InhibitorSelect™ Library I–III). Pipetting was carried out automatically by the ECHO®500 (Labcyte) to distribute volumes of small molecules into master plates. All working concentrations of 2, 10, and 50 µM, were further diluted from one master plate per experiment for higher accuracy using the Tecan Freedom Evo (Tecan) with a 96-head and directly applied to embryos at 10 hpf at a final volume of 100 µl per well to start the treatment. Propylthiouracil was added at a final concentration of 0.003% to each well at 22 hpf to prevent pigmentation of the embryos.

Pulse experiments started from tail bud stage (10 hpf), small molecules were washed out after four hours and embryos were grown under normal conditions until 36 hpf to analyze their phenotypes.