Chicken Organ Dissection and Calcium Imaging

P0 Gallus domesticus chickens were dissected as described previously [28] (link), [29] (link); the tectorial membrane was removed with subtilisin Carlsberg (Sigma Type XXIV) protease (50 µg/ml for 15 min). Extracellular solution was used at room temperature for all dissecting, loading, and imaging steps and contained the following (in mM): 87 NaCl, 0.5 KCl, 0.5 CaCl₂, 1.25 NaH₂PO₄, 2 ascorbate, 2 creatine, 6 Na-pyruvate, 75 sucrose, 25 D-glucose, 10 HEPES (pH 7.4, 310–320 mOsm). Fluo-4 AM dye was prepared as described in Figure 1, using the following reagents: Fluo-4 AM (Invitrogen, F14201), DMSO (Invitrogen, C6667), Pluronic F-127, 20% solution in DMSO (Invitrogen P3000MP). Bringing all dye reagents to room temperature before beginning was essential; moreover, all preparation and loading steps were carried out at room temperature in foil-wrapped tubes to protect dye fluorescence. To normalize for dye loading in fluid-jet experiments, the cell-permeable inert dye CellTracker Red CMTPX (CT-Red; Invitrogen C34552) was added to the organs for 15 min following Fluo4-AM loading. There was no obvious difference in the Fluo-4 signal between tall and short hair cells, and both cell types were analyzed in the group data.