Immunolabeling of Heteroxylan in P. ovata

The protocol from Phan et al. (2016) (link) was adapted for examination of HX in P. ovata mucilage. In brief, seeds were initially imbibed in 1X PBS for 30 min at room temperature, followed by a 60 min incubation in a 10-fold diluted primary LM11 (McCartney et al., 2005 (link)) or CCRC-M110 antibody with gentle agitation. The monoclonal CCRC-M110 antibody raised against Phormium tenax xylan was shown to bind P. ovata HX following the assay described in Pattathil et al. (2010) (link). Samples were washed in 1X PBS (5 × 1 min) and subsequently incubated for 60 min in a 100-fold dilution of goat anti-rat IgM conjugated with DyLite 550 (Thermo Fisher, USA) with mild agitation. Samples were again washed with 1X PBS (5 × 1 min). Whole seeds were individually mounted in 1X PBS on single cavity microscopy slides and images taken using a Zeiss M2 AxioImager with Zeiss filter set 43 (excitation BP 545/25, beam splitter FT 570, emission BP 605/70) and an AxioCam Mrm black and white camera. Images were processed using ZEN 2012 software (Zeiss, Germany).