2.6 Immunolocalization of a branched pectin epitope
Immunolocalization of branched 1-4 galactan with the LM26 monoclonal antibody (PlantProbes, Leeds, UK), was performed in the searcher stems, but not in twining stems, because they were much stiffer and embedded poorly or when embedded were difficult to section. Segments of 0.5cm from flexible branches were fixed with 4% w/v acrolein (Polysciences, Warrington, PA, USA) in a modified piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES) buffer adjusted to pH 6.8 (50 mM PIPES and 1mM MgSO4 from BDH, London, UK; and 5mM EGTA) for 24 h, then washed and dehydrated through a series of increasing aqueous acetone concentrations, one hour each: 10%, 30%, 50%, 70%, 90%, 100%. After that, they were incubated in a solution containing the Technovit 8100 for two weeks, and then hardened in anoxic conditions at 4ºC. Longitudinal and transverse 4µm sections were obtained with a Leica EM UC7 ultramicrotome (Leica Microsystems, Wetzlar, GE), mounted onto superfrost slides, and then used for immunolocalization. Pre-incubation with 5% bovine albumin serum (BSA), and three washes in 1XPBS, were followed by incubation with the LM26 monoclonal antibody for 1h. After washing the primary antibody with PBS, an anti-rat alexa488 secondary antibody, with a fluorescein isothianate marker-FITC was applied for 1h. Samples were then washed and observed with a Leica DM2500 microscope equipped with epifluorescence and a Leica DM600 camera, combining the 405 filter for autofluorescence with the 488 filter for the specific signal of the FITC.