Ion Currents at Cornea Wounds Figure 2. Measuring flux of specific ions at corneal wounds. A. Mounting eyes for wounding and probe measurements. A custom-made dish with wire loops holds the eye, leaving the cornea free for measurements and wounding (scale bar 2 cm). Right panel shown wound on cornea stained with fluorescein. B. For measurements, the cornea faces the electrode in the center of the dish. The eye can be rotated and/or the electrode moved to measure at different positions. C. Schematic showing wounded rat cornea with ion selective microelectrode in reference position (‘ref’) and measuring position (‘meas’) at the wound edge. Insert on right shows raw data recording trace (see Methods for details). D, E. Self-referencing mode. D. The electrode moves at low frequency (0.3 Hz) between two points (‘near’ and ‘far’) 30 mm apart at the wound edge. E. Recording trace which has been converted into ion concentration using the calibration trace (see below). F, G. Calibration. F. Calibration of the probe in standard solutions. G. A linear trendline obtained by plotting the amplifier output in mV vs. the logarithm of the ion concentration yields a formula (shown in red) used to calculate ion concentration and, in turn, the actual ion flux. doi:10.1371/journal.pone.0017411.g002 dessicant (Drierite; W.A. Hammond Drierite Co. Ltd, Xenia, OH, USA). To prepare an electrode for use, it was first back-filled using a disposable plastic Pasteur pipette (Phenix Research Products, Candler, NC, USA; cat # PP-137030) heat-pulled to a fine filament, with a 1 cm length of solution containing 100 mM of the ion to be measured. For example, NaCl is used if sodium or chloride are to be measured, CaCl2.2H20 for calcium, KCL for potassium, pH 7 buffer for protons. Pressure was applied via a PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e17411