A0855

The basal region of shoot explants was manually sectioned with a razor blade and immediately fixed in freshly prepared 3% 1-ethyl-3-(dimethylaminopropyl)-carbodiimide hydrochloride (EDAC) (Sigma-Aldrich) in 1× PBS containing 0.1% Triton X-100 (PBS-T) for 1 h on ice.
The sample sections were post-fixed for 1-2 h in 4% paraformaldehyde in PBS-T and washed three times in PBS-T for 5 min. The sample sections were then dehydrated and rehydrated with 30%, 50%, 70%, 100%, 70%, 50%, and 30% methanol in PBS-T (5 min each step) and incubated with 2% cellulase Onozuka R-10 (Duchefa Biochemie) for 30 min at 25 °C. After washing as above, sections were incubated overnight at 4 °C with a 1:100 dilution of the anti-IAA-C-monoclonal antibody (A0855; Sigma-Aldrich) in PBS-T. The sections were then rinsed three times for 10 min with PBS-T. Samples were incubated with 1:200 dilution of Alexa Fluor 647-conjugated donkey antimouse IgG (ThermoFisher Scientific) antibody for 2 h at 25 °C. The prepared samples were observed with LCSM as described elsewhere (Bustillo-Avendaño et al., 2018) .

For immunolocalization of IAA, root tips from primary roots of 2week-old plants were fixed in 4% paraformaldehyde and cells were digested with 0.2% driselase and 0.15% macerozyme for 1 h at 37°C, followed by membrane permeabilization with 3% IGEPAL CA630 and 10% DMSO for 1 h at 37°C. Further immunolocalization was performed as described in Lu et al. (2015) using primary anti-IAA antibody (1:200, A0855-Sigma-Aldrich) and anti-mouse IgG (whole molecule)-FITC Ab (1:1000, F0257, Sigma-Aldrich); images were taken using a TCS SP2 confocal microscope (Leica) with 495 nm excitation and 510-530 nm emission. Fluorescence intensity (FI) was measured using Ima-geJ software as described in Sharma et al. (2015) in which FI = integrated density À (area selected 9 mean background fluorescence). Relative FI was then calculated with respect to the wild-type.