Fig. 2. Use of salicylate-selective microelectrodes for measurement of the concentration of salicylate in droplets of fluid secreted by isolated Malpighian tubules in the Ramsay assay. An isolated pair of Malpighian tubules is placed in a droplet of bathing saline under paraffin oil. One Malpighian tubule remains in the saline, and the other is pulled out and wrapped around a stainless steel pin embedded in the Sylgard-line base of a Petri dish. Secreted fluid droplets collect at the ureter which is positioned just outside the bathing saline droplet. Secreted fluid droplets are collected on glass rods and placed on the bottom of the dish adjacent to calibration droplets containing known concentrations of salicylate in Drosophila saline. For each droplet, the potential difference between the salicylate-selective microelectrode (Sal ME) and the reference microelectrode is measured by a unity gain high impedance (>10¹⁵ ) operational amplifier. Voltages are digitized and recorded on a PC-based data acquisition system. Salicylate concentration in the secreted droplets is calculated from the voltage difference between the secreted droplet and the calibration droplets, as described in the methods.