Immunolocalization of PIN Auxin Transporters

Immunolocalization was performed as described (Kitakura et al., 2017 (link)). Briefly, seedlings were fixed with 4% paraformaldehyde in PBS (1h), adhered on MAS-coat slides (Matsunami glass, S9441), permeabilized by sequential treatment with 2.5% to 3.0% driselase (30 min at 37°C) and a mixture of 10% DMSO and 1% NP-40 substitute (1 h). 3% BSA in PBS was used for blocking and dilution of antibodies as follows: goat anti-PIN1 (1:400; Santa Cruz, sc-27163), rabbit andi-PIN2 (1:2000) (Abas et al., 2006 (link)), Cy3-conjugated secondary anti-rabbit (1:600; Sigma, C2306) and DyLight 649-conjugated secondary anti-goat (1:400; Jackson Immuno Research, 705-495-147) antibodies. After incubation with the antibody solutions, samples were washed with PBS and mounted in ClearSee solution (Kurihara et al., 2015 (link)). Confocal laser-scanning microscopy was performed with a Carl Zeiss LSM710 microscope.