Cellulose Localization in Hypocotyls

Fifteen-day-old hypocotyls were fixed overnight at 4 °C in 4% paraformaldehyde (Sigma-Aldrich, Steinheim, Germany) in phosphate buffered saline (PBS). After washing, sections of 5 mm were embedded in 5% agarose. Cross sections of 100 µM were cut using a vibratome (Leica Biosystems, Nussloch, Germany) and were stained with toluidine blue (Sigma-Aldrich), or used for cellulose immunodetection. The cellulose-binding module CBM3a probe (PlantProbes, Leeds, UK) specific for crystalline cellulose was diluted to 10 µg/mL in milk protein/PBS (MP/PBS, 5% w/v), incubated in mouse anti-His monoclonal antibody (1% in MP/PBS, Sigma-Aldrich) and finally incubated in 2% anti-mouse IgG coupled to fluorescein isothiocyanate (FITC) (Sigma-Aldrich) in MP/PBS. CBM3a incubation lasted for 1.5 h. Anti-His and IgG-FITC incubation lasted 1 h. Between each step, three washes of 5 min each with PBS were performed. The slides were mounted in 50% glycerol (Sigma-Aldrich) and observed with a confocal microscope LSM 510 Meta (Zeiss, Jena, Germany) with the following setting: excitation at 488 nm, filter HFT 488/594 and emission recorded with LP 505, as previously reported [19 (link)].