Citifluor af2 antifade reagent

To observe carbohydrate epitopes at root surfaces, 1 to 2 cm regions of whole barley roots with root hairs (WT) or equivalent regions of brb roots were excised and placed overnight in a 4% (w/v) formaldehyde fixative and processed for whole mount immunofluorescence labeling procedures essentially as described (Jackson et al., 2012 ). After antibody incubations, intact root regions were mounted using Citifluor AF2 antifade reagent (glycerol suspension; Agar Scientific Stansted, U.K.) and examined using an Olympus BX-61 microscope with epifluorescence irradiation from a mercury lamp and with excitation (∼480 nm) and emission (∼510 nm) for fluorescein isothiocyanate (FITC) fluorescence detection. Images were captured using a Hamamatsu ORCA285 camera and Volocity software. For each antibody, a manual exposure time was maintained for the capture of all shown micrographs across the two barley genotypes.