Fluorescence Quantification of AtRGS1-YFP

Fluorescence quantification for AtRGS1-YFP internalization was performed as described by Urano et al. (2012) (link) and Fu et al. (2014) (link). Sterilized, stratified seeds were germinated in 6-well plates containing 2 ml 1/2 × MS liquid medium (pH adjusted to 5.75 with 5 N KOH) at 23°C under darkness. Seedlings (7-day-old) were treated with 0 or 3% D-glucose (w/v) for 30 min. Hypocotyl epidermal cells located 2–4 mm below the cotyledon were imaged (Z stacks obtained) using a Zeiss LSM710 confocal laser scanning microscope equipped with a 20× Plan-NeoFluor (N.A. = 0.5) objective and a 40× C-Apochromat (N.A. = 1.20) water immersion objective. YFP fluorescence was excited by a 514 nm argon laser and detected at 526–569 nm by a photomultiplier detector. At least 10 sets of images from 5 seedlings were obtained for internalization quantification analysis by ImageJ software.