Propidium iodide staining and confocal microscopy

Four-day-old dark-grown hypocotyls were immersed in 0.2 mg/ml propidium iodide (Sigma) in water for 10 min and washed with water prior to imaging. Hypocotyls were placed between glass slide and coverslip separated by 400 μm spacers to prevent tissue crushing. Images were collected using a Leica TCS SP8 confocal microscope. Propidium iodide excitation was performed using a 552 nm solid-state laser and fluorescence was detected at 600-650 nm.