Sc 27163

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.

Immunostaining of PIN1 and PIN2 in Col-0 WT and cmi1 mutant roots was carried out essentially as previously described [74 (link)]. Primary antibodies used in this study were anti-PIN1 (1:1,000; sc-27163; Santa Cruz Biotechnology) and anti-PIN2 (1:400; N782248; NASC). Anti-rabbit Cy3 (1:600; CALTAG Laboratories, Invitrogen) and AlexaFluor 488 anti-rat (1:600; Invitrogen) were used as secondary antibodies. Fluorescence was observed using a Zeiss LSM780-NLO confocal microscope/multiphoton microscope. Cy3 was observed by excitation at 543 nm and emission at 560 nm and AlexaFluor by excitation at 488 nm and emission at 499–519 nm. Quantification of PIN2 relocation was performed by scoring the number of cells with different PIN polarities.