Citifluor glycerol pbs

Resin sections were used for immunofluorescence detection of cell wall epitopes. PBS with 5% (w/v) milk protein was added for 30 min at room temperature to prevent non-specific binding. Primary antibodies were used at a 1:5 dilution (except for directly coupled LM19, see below, which was used at 1:1000) in 5% milk/PBS for 1.5 h. Goat anti-rat, anti-mouse or ant-rat IgG Alexa Fluor488 (Life Technologies) were used as secondary antibodies in a 1:100 dilution in 5% milk/PBS and samples were incubated for 1 h. Calcofluor White (Sigma–Aldrich) was used at 0.02 mg/ml in PBS for 5 min for visualization of cell walls. Anti-fade reagent Citifluor glycerol/PBS (Agar Scientific) was used to mount slides. Immunofluorescence imaging was performed using an Olympus BX61 microscope (http://www.olympus-global.com/) equipped with epifluorescence irradiation. Micrographs were obtained with a Hamamatsu ORCA285 camera (Hamamastu, http://www.hamamatsu.com) and PerkinElmer Volocity software. All related and comparative micrographs were captured using equivalent settings, and relevant micrographs were processed in equivalent ways for the generation of datasets. In all cases the area shown in each micrograph is representative of the fibre tissue seen in at least 3 sections analysed from one plant. In the case the FM966 line the micrographs are representative of the analysis of more than 3 plants.