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.