Goat anti mouse 18 nm gold conjugated secondary antibody

A 2-mm region at the base of stems at growth 6.9 were chemically fixed, dehydrated, and embedded in LR white according to the method outlined in Wilson and Bacic (2012) (link). Thin sections (∼80 nm) were acquired for antibody labeling and post-stained (Wilson and Bacic 2012 (link)). For antibody labeling, samples were incubated with anti-6x-His tag monoclonal (Invitrogen, # MA1-21315) at 1:100 dilutions for 1 h at room temperature, and then overnight at 4 °C. Samples were then washed and incubated with goat anti-mouse 18 nm gold-conjugated secondary antibody (Jackson Immuno Research #115-215-166) at 1:20 dilutions for 1 h at room temperature. Detection of ultrastructure, HIS-YFP-FLA11, and HIS-YFP-FLA12 subcellular location was performed on 2 biological replicates using 2 technical replicates. Grids were imaged using a Jeol 2100 EM equipped with a Gatan Orius SC 200 CCD camera. Gold signals were quantified manually from TEM images and data were shown as mean ±Sd.

The protocol for preparation of plant cells for TEM was adapted from Wilson and Bacic (2012) (link). Stems embedded in LR White were sectioned using a Leica UC7 Ultramicrotome (Leica Microsystems, Germany) to a thickness of 90 nm. Sections were collected on formvar coated copper grids (Microscopy Solutions, Australia). Grids were stained using a mouse anti-6x-His tag primary antibody (Thermo Fisher #14-6657-80) diluted 1:100 (control grids with no primary antibody were also prepared), and 18 nm goat anti mouse 18 nm gold conjugated secondary antibody (Jackson ImmunoResearch #115-215-146) at 1:10 dilution. Grids were post-stained using 2% uranyl acetate for 5 min and Reynold’s lead citrate for 1 min.
The grids were viewed using a Joel JEM-2100 transmission electron microscope equipped with a Gatan Orius SC 200 CCD camera. Image analysis was performed with ImageJ software to measure gold density. The amount of gold per 1 μm² was measured as the total number of gold in the cytoplasm or cell wall divided by the area. For PM, 1 μm² was calculated as the area 0.05 μm either side of the PM. Two independent pFLA16:FLA16-VH transformed lines with two biological replicates each were investigated.