Five days old seedlings were incubated in 1 ml liquid growth medium (0.5x MS medium, 1% sucrose, pH 5.8) containing 20 μM Estradiol (Sigma Aldrich) for 5-6h at plant room conditions in 24-well cell-culture plates. Incubation was stopped by fixation with 4% paraformaldehyde in MTSB. Immunofluorescence staining was performed as described [49 (link)] or with an InsituPro machine (Intavis) [50 (link)].
Antibodies used: rat anti-tubulin 1:600 (Abcam), rabbit anti-AtγCOP 1:1000 (Agrisera), rabbit anti-KNOLLE 1:2000 [49 (link)], rabbit anti-clathrin (1:600) [51 (link)]. Alexa633 (Invitrogen) or Cy3-conjugated secondary antibodies (Dianova) were diluted 1:600.
Live-cell imaging was performed with 2 μM FM4-64 (Invitrogen, Molecular Probes).
Estradiol induction was performed using 20μM Estradiol for 5-6h at plant room conditions in 24-well cell-culture plates.
Antibodies used: rat anti-tubulin 1:600 (Abcam), rabbit anti-AtγCOP 1:1000 (Agrisera), rabbit anti-KNOLLE 1:2000 [49 (link)], rabbit anti-clathrin (1:600) [51 (link)]. Alexa633 (Invitrogen) or Cy3-conjugated secondary antibodies (Dianova) were diluted 1:600.
Live-cell imaging was performed with 2 μM FM4-64 (Invitrogen, Molecular Probes).
Estradiol induction was performed using 20μM Estradiol for 5-6h at plant room conditions in 24-well cell-culture plates.
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