Plan apochromat 20x 1.4na objective

Confluent organoids were harvested for imaging as follows. Media was removed, and following extensive washing in PBS, Matrigel domes were fixed in 4% PFA in PBS for 60 minutes, with periodic swirling to release whole organoids from Matrigel. A cut p1000 tip was used to transfer whole organoids to a tube, PFA was aspirated, and whole organoids washed several times in PBS. Organoids were permeabilized in perm/block buffer (5% donkey serum, 0.5% Triton X-100 in PBS) for 3 hours at room temperature. Organoids were then incubated with primary antibody (see Table 2 for antibody list) overnight at 4°C. The following day, organoids were washed extensively, and incubated with secondary antibody at room temperature for 3 hours. In the final 10 minutes, 200 µL of an 1 µg/mL DAPI solution was added. Organoids were washed extensively, and moved to chamber-well slides for imaging. Z-stacks were captured on a laser scanning confocal microscope (LSM700, Carl Zeiss) at 8-bit with Plan-Apochromat 20X/1.4NA objective using Zen software (RRID: SCR_013672).

β‐glucuronidase activity was carried out at 37°C overnight (16 hr) using 2 mM ferri/ferrocyanide as described (Weigel & Glazebrook, 2002). After GUS staining, whole seedlings were cleared and mounted on 50% glycerol and detected by light microscopy using differential interference contrast (DIC) on a Zeiss Axio Imager M1 (Carl Zeiss, Göttingen, Germany) microscope using a Plan‐Apochromat 20X/1.4 NA objective. Whole‐mount in situ hybridizations for miR390 were performed exactly as described (Dastidar et al., 2016). Confocal laser scanning microscopy was performed throughout the study using a Plan‐Apochromat 20x, 0.8‐NA lens on a Leica SP8 or SPE microscopes. Roots were stained with 10 μg/ml propidium iodide for 2 min, rinsed, mounted in water, and visualized after excitation by an argon 488‐nm laser line. The fluorescence emission was collected from 590 to 700 nm (propidium iodide) and 496 to 542 nm (GFP).