Hemp hypocotyls were embedded in Technovit 7100 resin (Kulzer). Briefly, sections of 5 mm were fixed in glutaraldehyde/paraformaldehyde/caffeine (1%/2%/1% v/v in Milli-Q water) under vacuum for 15 min and 24 h at 4°C, dehydrated in an ethanol series (70–95–100%), impregnated in resin containing PEG 400 (2% v/v) and dimethacrylate ethylene glycol (0.4% w/v) and finally included. Cross sections of 10 μm thickness were cut using a microtome (Leica) and stained with toluidine blue or used for immunohistochemistry (IHC). Image acquisition was performed with a Leica DMR for toluidine blue and with a confocal microscope LSM 510 Meta (Zeiss) for IHC.
LM5 (β-1,4-galactan), LM10 (xylan) and LM15 (xyloglucan) (Plant Probes) antibodies were diluted 10-fold in milk protein (MP)/PBS (5% w/v). Sections were then incubated for 1.5 h, rinsed three times in PBS and incubated for 1.5 h with the anti-rat IgG coupled to FITC (Sigma) diluted 100-fold in MP/PBS. Before observation, three washing steps with PBS were performed. CBM3a (crystalline cellulose, Plant Probes) was diluted to 10 μg/mL in MP/PBS, incubated in mouse anti-His monoclonal antibody (1% in MP/PBS, Sigma) and finally incubated in 50-fold diluted anti-mouse IgG coupled to FITC (Sigma). Each incubation lasted for 1.5 h. Between each step, three washes with PBS were performed. The slides were mounted in Möwiol 4-88 (Sigma) and observed with the following settings: excitation at 488 nm, filter HFT 488/594 and emission recorded with LP 505. The microscope settings were kept rigorously constant between the different observations for a given epitope. Negative controls where either the primary or secondary antibody was omitted resulted in a very weak and negligible signal.
LM5 (β-1,4-galactan), LM10 (xylan) and LM15 (xyloglucan) (Plant Probes) antibodies were diluted 10-fold in milk protein (MP)/PBS (5% w/v). Sections were then incubated for 1.5 h, rinsed three times in PBS and incubated for 1.5 h with the anti-rat IgG coupled to FITC (Sigma) diluted 100-fold in MP/PBS. Before observation, three washing steps with PBS were performed. CBM3a (crystalline cellulose, Plant Probes) was diluted to 10 μg/mL in MP/PBS, incubated in mouse anti-His monoclonal antibody (1% in MP/PBS, Sigma) and finally incubated in 50-fold diluted anti-mouse IgG coupled to FITC (Sigma). Each incubation lasted for 1.5 h. Between each step, three washes with PBS were performed. The slides were mounted in Möwiol 4-88 (Sigma) and observed with the following settings: excitation at 488 nm, filter HFT 488/594 and emission recorded with LP 505. The microscope settings were kept rigorously constant between the different observations for a given epitope. Negative controls where either the primary or secondary antibody was omitted resulted in a very weak and negligible signal.
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