Non-fucosylated xyloglucan-5 (clone CCRC-M48)
AS16 3221-1ml | Clonality: monoclonal | Host: Mouse | Reactivity: Arabidopsis thaliana, Camelina sativa, Tamarindus indicus
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Localization of non-fucosylated xyloglucan-5 (red) in Arabidopsis thaliana hypocotyl, Calcufluor White counterstain (blue) and cell wall autofluorescence (yellow).
The 31 days-old hypocotyls were immersed in 150 μL PME fixation buffer (25 mM PIPES, 1 mM MgSO4, 1 mM EGTA) and then subjected to three consecutive cycles of 5 min-long vacuum infiltration (21°C, 68 kPa). Afterwards they were washed three times in PME (21°C, 68 kPa) prior to storage at 4°C in PME. Hypocotyls were encased in 1 cm3 blocks of 5% agar at 65°C, and stored at 4°C to set. Transverse 40 μm thick sections were cut from segments using a VT100S vibrating microtome (Leica) and blocked for at least 1 h in 5% bovine serum albumin in TBST. Blocking solution was discarded and sections were incubated at 4°C for 16 h with 5 μl of the anti-Non-fucosylated xyloglucan-5 antibody, followed by 2 washes in 100 μL TBST. Sections were then incubated for 1 h at 21°C in the dark in 10 μl of 2 μg/μl Alexa FluorTM 568 donkey anti-mouse IgG (H+L; 1:36). Sections were again washed twice in 40 μL TBST prior to counter-staining with 0.015% Calcofluor White (Sigma-Aldrich). Sections were again washed twice in 100 μL TBST to remove excess counter-stain and unbound secondary antibody. Immunofluorescence of AlexaFluor 568 was excited with a 561 nm laser, and emitted light filtered at 575–600 nm, while Calcufluor White was subsequently scanned on an independent channel with a 405 nm laser and emission observed at 420–430 nm using laser scanning microscope Zeiss LSM780 point-scan system at 1024 × 1024 pixels (pixel size, 0.6–0.83 μm) with a 10X objective.
Courtesy Dr. Urs Fisher, Umeå Plant Science Centre, Sweden