2.5 Immunolocalization of pectin and arabinogalactan glycoprotein epitopes
A preliminary test with the general dye Ruthenium red was used in freshly dissected leaves (Supplementary Figure 1) to evaluate the presence of neutral pectins (Colombo & Rascio, 1977). Similarly, a preliminary test for arabinogalactan protein (AGP) presence was applied to cleared leaves with a 2% solution of the chemical reagent beta glucosyl Yariv reagent in 0.15M NaC1 (Yariv, 1967). The β-GlcYR reacts with arabinogalactan proteins giving a red color upon precipitation (Supplementary Figure 2).
To identify the presence of pectic and AGP epitopes in the leaves ofCapparis odoratissima , two monoclonal antibodies that detect highly un-esterified homogalacturonans (JIM5) and AGPs (JIM8) (Carbosource services, Georgia, USA) were used. Transverse and paradermal sections (4 µm thick) of embedded leaves were incubated with 1XPBS 3x for five minutes each, followed by 5% bovine albumin serum (BSA) for five more minutes, and finally the undiluted primary monoclonal antibody for 45 min. The sections were then washed three times in 1XPBS, five minutes each, and incubated again with an anti-Rat secondary antibody Alexa 488 conjugated with a fluorescent compound Fluorescein Isiothianate for one hour. Three final washes with 1XPBS preceded observations with a confocal microscope. Negative controls were treated in the same way, but substituting the primary antibody with a solution of 1% BSA in PBS.