Benzamil hydrochloride hydrate

Fish were treated with cadmium chloride hemipentahydrate (Thermo Fisher Scientific, Waltham, MA, USA) dissolved in EM for 3 h at 28.5°C. Some fish were additionally treated with benzamil hydrochloride hydrate (Sigma-Adrich, St. Louis, MO, USA), zinc sulfate heptahydrate (Sigma-Adrich, St. Louis, MO, USA), or copper sulfate pentahydrate (Sigma-Adrich, St. Louis, MO, USA). A stock of benzamil was made at a concentration of 40 mM in dimethyl sulfoxide (DMSO, Sigma-Adrich, St. Louis, MO, USA). Control animals for the benzamil experiment were therefore treated with DMSO diluted in EM at a 1:200 ratio to match the DMSO levels in the benzamil group. Stocks of all other compounds were made in water and DMSO was not used for those experiments. For all treatments, fish were put into six well plates containing net-well inserts and the net-wells were moved to plates containing the different solutions the fish were exposed to. Following treatment, animals were washed three times in EM, euthanized with MS-222 and immediately fixed for immunostaining.

Lysine fixable 3 kDa and 10kDa-FITC or Texas Red-labeled dextrans were purchased form ThermoFisher scientific Inc (Waltham, MA.). Dextrans were diluted in Hanks' balanced salt solution without Ca²⁺ and Mg²⁺ (HBSS-CFM) at a final concentration of 10 mg/mL, aliquoted in black opaque tubes and stored at −30°C until use. As indicated by the manufacturer, the actual molecular weights of dextran molecules in each preparation has a distribution, and contains polymers with molecular weights ranging from 1.5 to 3.0 kDa (including the dye) in the case of 3 kDa dextran-TR, and 50% of the dextran molecules contains a dye, on average.
Hank’s balanced salt solution (HBSS), HBSS-CFM, phosphate buffered saline (PBS), Dulbecco’s Modified Eagle Medium (DMEM), DMEM media without calcium, and Leibovitz’s L-15 medium with GlutaMAX were also purchased from ThermoFisher scientific Inc. Cell impermeant BAPTA tetrapotassium salt (Molecular Probes, ThermoFisher scientific, Inc.) was dissolved at 50 mM in water. A stock solution of 30 mM benzamil hydrochloride hydrate (Sigma-Aldrich, St. Louis, MO) was prepared in DMSO. Amiloride hydrochloride (EMD MILLIPORE, Burlington, MA) was dissolved at 150 mM in DMSO. Aminoglycosides, dihydrostreptomycin sesquisulfate, and neomycin trisulfate salt hydrate (Sigma-Aldrich) were dissolved at 50 mM in water.