Histochemical GUS staining, GFP, and FRET experiments
Histochemical GUS staining, GFP microscopy and FRET were performed as
previously described with some changes
(Datta et al., 2006;
Hou, Wu, & Gan, 2013;
Zhang, Zhang, et al., 2014).
For GUS staining assay, the young seedlings (4 days after germination)
were fixed and incubated in GUS-staining solution for 24 h at 37°C.
Stained samples were then cleaned with 75% ethanol and observed by
dissecting microscope.
For
GFP assay, the fusion COL13 -GFP constructs were transformed into
protoplast for transient expression as previously described
(F.-H. Wu et al., 2009). Ten stable
transgenic plants with COL13 -GFP were obtained by the floral-dip
method. The photos of GFP were taken by
confocal microscope Olympus.
For FRET assay, images were acquired using an Olympus confocal
microscope, protoplasts were visualized 16 h after transforming. The CFP
was excited by a laser diode 405 laser and the YFP by an argon-ion
laser. The target regions were bleached with 100 iterations by the
argon-ion laser at 100%.