Measuring Larval Midgut pH Dynamics

pH was measured in the larval midgut using Thymol blue, m-Cresol purple, Cresol red, Bromocreosol purple, BromoThymol blue, Phenol Red and Congo red indicator dyes (all purchased from Sigma- Aldrich). Indicators were added to melted Drosophila diet (0.1% w/v), immediately mixed, and allowed to cool to room temperature. Larvae of the appropriate genotype were added, and after 2 hours the midgut excised in Schneider’s insect medium (Invitrogen). Micrographs were taken immediately using a Sony CyberShot NEX-C3 mounted on a Leica stereo microscope, as pH remains stable for only a few minutes after dissection. Images were processed using Adobe Photoshop CS5.1. Where noted, the diet was also supplemented with 1 mM Omeprazole (Sigma-Alrdich) or 100 μM acetazolamide (Sigma-Aldrich).