Goat anti rabbit igg antibody conjugated with 10 nm gold

Ultrathin sections were prepared and immunogold labeled according to the protocol described by Zechmann and Müller (2010) (link). Briefly, the ultrathin sections from J and A leaves were initially incubated with the primary antibody (anti-glutathione rabbit polyclonal IgG, Agrisera Corp., Vännäs, Sweden) at a dilution of 1:3,000 in the dilution buffer. This antibody recognizes only the conjugated glutathione and GSH but not GSSG. Then the ultrathin sections were treated with secondary antibody (goat anti-rabbit IgG antibody conjugated with 10 nm gold, Sigma-Aldrich, St. Louis, MO, United States) at a dilution of 1:20. The sections were finally double-stained with uranyl acetate-lead citrate and examined with a JEM-100S electron microscope (JEOL, Tokyo, Japan). Micrographs of randomly photographed immunogold labeled sections were digitized with computer aided drafting software and gold particles were manually counted. A minimum of at least 10 different cells were analyzed for gold particle density per sample. The gold particle density data in the organelles were normalized against the cytosolic background.

Proteins were extracted and CWIN activity was assayed, as previously reported^{35 (link)}. For the immunogold labeling assay, litchi funicles were cut into 2–3 mm³ cubes that were immediately fixed with 4% (w/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 100 mM precooled PBS (8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 3 mM KCl, and 500 mM NaCl, pH 7.2) for 4 h. Dehydration and subsequent infiltration was conducted according to Zhang et al^{36 (link)}. Ultrathin sections (~50 nm) were cut and mounted on 100-mesh nickel grids coated with 0.3% Formvar films for subsequent immunolabeling. The ultrathin sections were first blocked by floating the grids on droplets of PBS (pH 7.4) supplemented with 50 mM glycine for 30 min at room temperature and continuously blocked with PBS (pH 7.4) supplemented with 0.1% (w/v) gelatin, 0.5% (w/v) bovine serum albumin (BSA), and 0.1% (v/v) Tween 20. Antiserum against CWIN was custom made in Bioleaf Biotechnology Co., Ltd (Shanghai, China). Goat anti-rabbit IgG antibody conjugated with 10 nm gold was purchased from Sigma.