Immunogold Labeling of Glutathione

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.