Arabidopsis leaves were fixed in a solution of 4% (v/v) paraformaldehyde and 0.5% (v/v) glutaradehyde in 0.1 M phosphate buffer (pH 7.2) for 20 min under vacuum and then a further 3 h at room temperature.
The specimens were then dehydrated in a graded ethanol series, infiltrated in stepwise increments of LR white resin (London Resin, Theale, Berkshire, UK) and polymerized at 45 °C for 24 h.
Materials for immuno-gold labelling were prepared according to the procedure of Varagona and Raikhel (1994) with the modification as described.
Specimens (90 nm) were sectioned using a Leica Reichert Ultracut S microtome and mounted on formvar-coated slotted grids.
Grids were incubated in a blocking solution of TTBS containing 1% (w/v) fish skin gelatin and 1% (w/v) BSA for 30 min.
Anti-ACBP4 antibodies diluted 1:50 in blocking solution were added and incubated at room temperature for 2 h.
The grids were then rinsed three times, each for 5 min, in TTBS and then incubated with 10 nm gold-conjugated goat anti-rabbit IgG secondary antibody (Sigma), diluted 1:20 with blocking solution.
Grids were rinsed three times, each for 5 min in TTBS, following by three 5-min rinses in distilled water.
After being stained in 2% (w/v) uranyl acetate for 6 min followed by 2% (w/v) lead citrate for 6 min, the sections were visualized and photographed using Philips EM208s electron microscope operating at 80 kV.
Controls were performed excluding the primary antibody.
(B, C, D) Immuno-gold labelling of ACBP4 in an Arabidopsis leaf cell using transmission electron microscopy.
Transverse sections were stained with affinity-purified ACBP4-specific antibodies.
(B) Transverse sections of leaves stained with ACBP4-specific antibodies.
(C) Magnification of the boxed area in (B).
(D) Control labelling of a leaf cell using secondary antibodies alone.
Arrowheads, gold particles.
V, vacuole; C, cytosol; Ch, chloroplast; N, nucleus; Cw, cell wall; Bars in (B) represent 2 μm, and in (C, D), 0.2 μm.
Immuno-electron microscopy was carried out using transverse sections of leaves of 2-week-old Arabidopsis germinated and grown in MS medium under a 16/8 h light/dark regime.
Although immuno-gold labelling with the anti-ACBP4 antibodies was mostly evident in the cytosol, some signals were detected at the periphery of the nucleus, (Fig.
3B, C).
In the control, when the primary antibody was replaced by blocking solution, no significant immuno-gold labelling was observed (Fig.
3D).
The immunolocalization of signals at the periphery of the nucleus may have culminated from the interaction of ACBP4 with AtEBP.
In this study, GFP:AtEBP was not confined to the nucleus but was also detected in the cytosol where it could interact with ACBP4.
ACBP4:DsRed, transiently-expressed in tobacco leaves, was predominantly targeted to the cytosol but immuno-electron microscopy indicated localization of ACBP4 in the cytosol with signals detected at the periphery of the nucleus, perhaps as a consequence of its interaction with AtEBP.