3 ml syringe with attached silicon tube to push the air-bubble at the tip out, forcing the liquid to the tip. The electrode was then tip- filled with a small amount (50–100 mm length) of ionophore specific for the ion to be measured by attaching the electrode to a micro-positioner and dipping the electrode tip into a droplet of ionophore under a dissecting microscope. Reference electrodes were glass capillaries as above, cut into 5 cm lengths and briefly fire polished in a bunsen burner flame. They were filled with 3 M of NaCl, K-acetate or KCl, with 2% agar. Agar was added to the solution of choice and brought to the boil on a hotplate to dissolve the agar. The hot solution was drawn into the electrode with a plastic Pasteur pipette and the electrode dropped into cold KCl, NaCl or K-acetate solution. Reference electrodes prepared thus can be stored under appropriate solution in Petri dishes for several weeks prior to use. Reference electrodes must not contain the ion to be measured. Reference electrodes were mounted in a straight microelectrode holder with a gold 2 mm male connector and Ag/AgCl pellet (WPI; cat # MEH3S) and mounted on a 3-dimensional micro- positioner (Newport Corporation, Irvine, CA, USA). Measuring electrodes were mounted in a straight microelectrode holder with a gold 1 mm male connector and Ag/AgCl wire (Warner Instruments, Holliston, MA, USA; cat # QSW-A15P) and attached to a headstage mounted on a computer-controlled electronic 3-dimensional micro-positioner (Newport). The mea- suring electrode and reference electrode were connected to an Ion Amp V3.0 amplifier (BioCurrents Research Center, Woods Hole, MA, USA). As well as static measurements of ion concentration (Fig. 2C; see section Selective ion flux measurements below), electrodes were also used in self-referencing mode, where the electrode moves between two points 30 mm apart at low frequency (0.3 Hz) near the wound edge [35,36]. The electrode movement was controlled by a computer running IonView32 software (BioCurrents Research Center) controlling the electronic 3-dimensional micro-positioner. The electrode pauses at each position (‘near’ and ‘far’) and the output is recorded to computer (Fig. 2D, E). If an ion flux is present, the electrode detects a difference in ion concentration between its two positions. The ion flux can be calculated using the formula: J = Cu(dc/dx) where C is the ion concentration in the solution, u is the ion mobility, and dc is the concentration difference over distance dx. Ion flux data are presented in pmol/ cm2/sec or nmol/cm2/sec. Data were recorded onto computer using IonView32 software. Calibration of electrodes Electrodes were calibrated in standard solutions that bracketed the concentration of ion present in the BSS solution (Fig. 2F). For example, BSS solution contains ,150 mM Na+, so the sodium electrode was calibrated in 10, 100 and 200 mM NaCl. Plotting the logarithm of the molar concentration against the output of the amplifier (in mV) usually gave a linear fit with an R2 value of at least 0.99 (Fig. 2G; R2 value of 1.0 indicates perfect fit of a linear trendline), and the formula describing the line was used to convert the mV readings from the amplifier during sample measurements into actual ion concentrations from which ion flux can be calculated. Selective ion flux measurements First, the eye was mounted in the chamber (containing BSS saline at room temperature) under the dissecting microscope and allowed to equilibrate to the new temperature. The reference and measuring electrodes were placed in the bath. With the electrode at reference (bath) position approximately 1 cm from the cornea, the background value was recorded for a few seconds. The Ion Currents at Cornea Wounds electrode was then moved into measuring position about 30 mm from the intact, unwounded cornea surface for 10–15 seconds until the new value was stable. The electrode was then moved back to reference position and the recording stopped when the stable background value was reached. The cornea was then wounded as above and measurements made at the left wound edge (Fig. 2C). This position was chosen since we previously showed that the current at the wound edge was significantly greater than at the wound center, indicating substantial ion pumping and/or leakage at the cut edge of the corneal epithelium [6]. This gives a single ‘peak’ (see Fig. 2C insert on right) and the difference between the background and the wound value in mV can be converted into the difference in ion concentration using the equation derived by plotting the calibration readings (Fig. 2G). Wound edge measurements were made at various time-points after wounding to give timelapse data of wound ion concentration change over time. We also did some long time-lapse experiments up to 6 hours. In self-referencing mode, the oscillating electrode was first placed near the intact cornea to measure unwounded ion flux, and after wounding placed near the wound edge and measurements made in that position for up to 90 min (Fig. 2D, E). Drug treatments Aminophylline (cat # A1755) and ascorbic acid (cat # A5960) were obtained from Sigma-Aldrich (St. Louis, MO, USA). We previously showed that drugs which affect ion flux also alter cornea wound electric current and rate of wound healing [6,34]. Here we tested the effect of aminophylline (10 mM) and ascorbic acid (1 mM) on calcium, sodium and chloride ion flux. Eyes were kept in BSS solution at room temperature with drug for 20 minutes before beginning experiments. We found that the presence of aminoph- ylline interfered with chloride ionophore I cocktail A, perturbing the electrode responses and giving unpredictable results. Therefore, for chloride flux measurements in the presence of aminophylline, we used a different ionophore: chloride-selective liquid ion-exchanger microelectrode cocktail A (Sigma-Aldrich; cat # 24899). Data Ion-selective micro-electrode data are expressed two ways. First we show the concentration difference between the reference position in the bath solution, and the measuring position close to the cornea surface or wound edge (see Fig. 2C). For example, Figure 3A shows calcium concentration at unwounded cornea (time 0) and at wound edge (wound edge has a relatively higher concentration). We then present data of actual ion flux, calculated from data recorded with the electrode in self-referencing mode as it oscillates close to the cornea surface (see Fig. 2D, E). For example, Figure 3B shows calcium flux before and after wounding, showing a steady increase of calcium efflux (outward ion flow) after wounding. For each ion measured, the concentration difference data, and ion flux data, correlated. For example, higher ion concentration at wound edge = ion efflux (outward flow). Note that in all timelapse graphs (Figs. 3–7), the measurements made at intact cornea surface before wounding are shown at time zero. Immunofluorescence Rat cornea tissue were fixed with 4% paraformaldehyde (Sigma-Aldrich) in phosphate-buffered saline, pH 7.4 (PBS; Sigma-Aldrich) for 20 min. Cryo-sections were cut and collected on gelatin-coated slides. Non-specific binding sites were blocked with PBS containing 2% bovine serum albumin (BSA; Sigma- Aldrich), 10% donkey serum plus 0.3% Triton X-100 (Sigma- Aldrich), for 30 min at room temperature. A monoclonal antibody against CLC2 (1:200, Santa Cruz Biotechnology, Inc., Santa PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e17411