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.