As09 633

Selected plant cell components were visualized by immunocytochemical reactions on resin-free sections with specific primary antibodies provided by Agrisera (AHB2, Cat. No. AS132 745) or PlantProbes (Univeristy of Leeds, Leeds, UK), as follows: JIM5 (Cat. No. JIM5), JIM7 (Cat. No. JIM7), JIM11 (Cat. No. ELD030), JIM20 (Cat. No. ELD033), JIM8 (Cat. No. ELD024), JIM13 (Cat. No. JIM13-050), LM5 (Cat. No. LM5-050), LM6 (Cat. No. LM6-050), LM8 (Cat. No. LM8), LM24 (Cat. No. LM24). Antibodies were diluted (1:20) in 1% BSA in 1× PBS buffer (pH 7.2). The sections were incubated overnight at 4 °C. Next, sections were washed 3 times with 1× PBS buffer (pH 7.2) and incubated with secondary antibodies (goat anti-rat conjugated with FITC, Abcam, Cambridge, UK; goat anti-rabbit IgG DyLight 488 conjugated against AHB2, AS09 633, Agrisera, Vännäs, Sweden), 1:500 diluted in 1× PBS buffer. All reaction steps were conducted following our optimized protocol [109 (link)]. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) according to the producer′s recommendation. The results were documented using a fluorescent microscope DM6000B (Leica Microsystems GmbH, Wetzlar Germany). Control reactions were performed by omitting the incubation step with primary antibodies (Supplementary Figure S2).

The AZ sections for immunofluorescence studies were prepared according to Wilmowicz et al. [5 (link)]. The sections on slides were blocked in 1% bovine serum albumin (BSA) for 2 h and incubated with primary antibody anti-ACC (AS11 1800, Agrisera) and anti-ABA (AS06195, Agrisera), and subsequent steps were performed according to the protocol described by Wilmowicz et al. [5 (link)]. For immunolocalization of MPK6 and CAT, the sections were incubated overnight at 4 °C in primary antibody solution prepared by dilution the primary antibody anti-MPK6 (AS12 2633, Agrisera) and anti-CAT (AS09 501, Agrisera) 1:25 in 1% BSA dissolved in 1× PBS (pH 7.2). A DyLight Alexa 488 conjugated IgG diluted 1:250 in PBS buffer for 2 h at 37 °C was served as the secondary antibody (AS09 633, Agrisera). Negative control reaction required for validation of the immunohistochemical findings was performed by omitting the incubation with the primary antibody, and showed no labeling (Supplementary Figure S3).