Zebrafish were maintained in standard conditions (Westerfield, 2000 ) at the University of Manchester Biological Services Unit according to the UK Animals Act 1986. The ocrl−/− mutant line (ZDB-GENO-120531–1) has been described previously (Ramirez et al., 2012 (link)). WT fish were of AB background.
Lysine-fixable 10-kD dextran labeled with Alexa Fluor 488 (Molecular Probes) was prepared in PBS at 2 µg/µl final concentration. Zebrafish embryos at 72 h after fertilization were treated for 60 min with DMSO control (0.005% DMSO), 5 µM m-3m3fbs, or 5 µM o-3m3fbs by addition to the water. Embryos were then anesthetized with 0.2 mg/ml MS222 (Sigma-Aldrich) in chorion water, and tracer was injected into the common cardinal vein using a glass micropipette PLI-90 Pico-Injector (Harvard Apparatus). Embryos were returned to the respective drug treatments and incubated at 29°C. Pronephric accumulation was assessed 2 h after injection on whole mount embryos using a fluorescent dissecting stereomicroscope (MZ10F; Leica).
Lysine-fixable 10-kD dextran labeled with Alexa Fluor 488 (Molecular Probes) was prepared in PBS at 2 µg/µl final concentration. Zebrafish embryos at 72 h after fertilization were treated for 60 min with DMSO control (0.005% DMSO), 5 µM m-3m3fbs, or 5 µM o-3m3fbs by addition to the water. Embryos were then anesthetized with 0.2 mg/ml MS222 (Sigma-Aldrich) in chorion water, and tracer was injected into the common cardinal vein using a glass micropipette PLI-90 Pico-Injector (Harvard Apparatus). Embryos were returned to the respective drug treatments and incubated at 29°C. Pronephric accumulation was assessed 2 h after injection on whole mount embryos using a fluorescent dissecting stereomicroscope (MZ10F; Leica).
