Histochemical staining of lignin

Stem pieces (5 mm-length) corresponding to internode 12–14 from the apical meristem were fixed with 1.25% (v/v) glutaraldehyde in methanol by the freeze substitution method [37]. Tissue pieces were embedded by immersing them sequentially into 100% methanol, 100% ethanol, 3:1 (v/v) ethanol: Histolemon (Carlo Erba, France), 1:1 (v/v) ethanol: Histolemon, 1:3 (v/v) ethanol:Histolemon, Histolemon and Histolemon: Paraplast X-tra (Leica, Germany). All steps were performed at room temperature and under continuous agitation. Finally, the Histolemon: Paraplast X-tra mix was replaced with pure liquid Paraplast X-tra at 62°C. Sections of 10 μm were cut using an RM2125 RTS manual microtome (Leica) and mounted on poly-L-lysine-coated glass slides (Menzel-Gläser, Germany). Sections were deparaffinized and hydrated sequentially through an ethanol series. Staining was performed with 1% phloroglucinol in 2:1 ethanol absolute:HCl and images were captured with an AZ100 Multizoom Microscope (Nikon, UK)