Tissue harvesting for microscopic analysis
    Stem segments bounded by paper tags were harvested from individual plants in sequence from top to bottom. Upon excision, segments were immersed in 150µL of fixation buffer (stock 2X PME; 50mM 2-[4-(2-sulfoethyl)piperazin-1-yl]ethanesulfonic acid (PIPES), 2mM MgSO4, 2mM  EGTA), within 0.2ml dome-cap thermal cycler tubes.  Segments were then subjected to three consecutive cycles of five minute vacuum infiltration at 20 inches Hg and washed 3 times in 1X PME prior to long-term storage at 4oC in 1X PME.  Segments were individually encased in 1cm3 blocks of 5% agar at 65oC, and stored at 4oC to set.  40µm-thick transverse sections were cut from segments using a vibrating microtome (Model VT100S, Leica), separated from agar encasement with a sable-hair ('00') brush, then blocked for at least one hour in 5% bovine serum albumin (BSA) in 1X TBST (10mM TRIS, 0.25M NaCl, 0.1% TWEEN).  
    
Immunolabelling and staining
    Sections were mixed to randomize developmental difference, randomly allocated from each biological replicate pool, along with 100µL of fresh blocking solution, to wells of 96-well plate (BD Falcon), grouping three biological replicates row-wise according to antibody or negative control. Antibodies included in this experiment are listed in Table S1 providing 96-well plate position, source animal, supplier, immunogen, epitope structure, and antigen; information derived from CCRC database, WallBioNet (glycomics.ccrc.uga.edu/wall2/index.html). These 55 mAbs  collectively provide broad coverage of the major cell wall glycan classes relative to the coverage offered by the entire set of available cell wall glycan-directed antibodies (Figure S3) most of which were previously described in literature.  Blocking solutions were swapped with 15µL 1:36 dilutions of supplied antibody solutions (see Table S1) using gel-loading tips, then sections were incubated at 4oC for 16 hours. Sections were washed two times in 100µL 1X TBST, then incubated one hour at 21oC in the dark in secondary antibody; either 15µL of 2µg/µL Alexa Fluor™ 488 donkey anti-rat IgG (H+L) (Invitrogen) or 2µg/µL Alex Flour™ 488 goat anti-mouse IgG (H+L) (Invitrogen) based upon the requirements of the primary antibodies (see Table S1).  Sections were again washed 2 times in 100µL 1X TBST, prior to counterstained with 0.015% Congo Red (Fluka, Buchs, Switzerland).  Sections were again washed 2 times in 100µL 1X TBST to remove excess counterstain and unbound secondary antibody.