As lmd laser dissection microscope

For localization of heteroxylan a protocol was adapted from Willats et al. (2001) (link). Mature seeds were hydrated in 1×PBS for 30 min with gentle shaking and incubated for 90 min in a 10-fold dilution of primary antibody, LM11 (McCartney et al., 2005 (link)). Samples were washed in 1×PBS (5 × 1 min) and incubated for 90 min in a 100-fold dilution of goat anti-rat IgG conjugated with Alexa Fluor^® 488 (Invitrogen, USA, A1100), washed as above and counterstained with 0.2 µg ml^–1 propidium iodide (Sigma-Aldrich, P417). Whole seeds were mounted in 1×PBS and imaged using a Leica AS LMD laser dissection microscope with attached DFC 480 camera.
For double labelling with CBM3a, samples were incubated with a 10-fold dilution of primary antibody LM11 and 100-fold dilution of secondary antibody goat anti-rat IgM conjugated with DyLight 550 followed by a 500-fold dilution of CBM3a and a 300-fold dilution of mouse anti-His antibody. The His probe was labelled with goat anti-mouse IgG conjugated with Alexa Fluor 488. All antibodies were applied for 60 min with gentle shaking and samples washed in 1× PBS (5 × 1 min) between each incubation.
For immunolabelling of thin sections, the protocol of Burton et al. (2011) was followed. Transmission electron microscopy was carried out using a Philips CM100 microscope (Adelaide Microscopy).

Ruthenium red staining followed the protocol from Arsovski et al. (2009) with modifications excluding pre-hydration and imbibition for 10 min. Imaging was conducted using a Zeiss Stemi 2000-C dissecting microscope with an attached AxioCam ERc 5s camera. For calcofluor white staining, mature seeds were imbibed in a solution of 0.002% (w/v) calcofluor white (Sigma-Aldrich, F3543) with 0.01% Triton X-100 (Sigma-Aldrich, T8532) in 100 ml Tris (pH 8.0) for 10 min and imaged using a Leica AS LMD laser dissection microscope with an attached DFC 480 camera. To confirm developmental stages, seeds were cleared as per Tucker et al. (2012) (link) using Hoyer’s light solution (Anderson, 1954 (link)) and observed using differential interference contrast (DIC) and Nomarski optics on a Zeiss M2 Axio imager. To observe anatomical details of seed coat development, staged seed samples were fixed in 0.25% glutaraldehyde, 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS; pH 7.2), embedded in LR-White resin and sectioned to 1.0 μm before staining in 0.01% toluidine blue in 0.1% sodium tetraborate as per Aditya et al. (2015) (link).

The images of these sections were taken with a Leica AS LMD laser dissection microscope with a DFC 480 camera at 10x zoom. A scale bar was provided with the images so that results of the RootAnalyzer algorithm can be converted from pixels to μm.