Tissue was incubated with 2% BSA (Sigma) in PBS for 30 min to block nonspecific binding sites. The tissue was incubated in a chicken polyclonal anti-GFP antibody (Abcam) in blocking buffer for 4 d at room temperature. Tissue was then incubated in biotin-conjugated goat anti-chicken IgY secondary antibodies for 1 h at room temperature. Sections were incubated with the ABC reagent (Vector) for 1 h, followed by a DAB peroxidase reaction. Sections were immediately processed for electron microscopy.
Stained tissue was postfixed with 4% osmium tetroxide for 1 h, dehydrated through graded alcohols, and polymerized in Epon between glass slides and coverslips coated previously with Liquid Release Agent (Electron Microscopy Sciences). Areas of interest were selected based on the DAB labeling and re-embedded in Epon. These re-embedded sections were cut using an ultramicrotome in 70-nm-thick ultrathin sections. These 70-nm sections were examined with a JEOL transmission electron microscope and photographed at primary magnifications of 3000–4000×.
Stained tissue was postfixed with 4% osmium tetroxide for 1 h, dehydrated through graded alcohols, and polymerized in Epon between glass slides and coverslips coated previously with Liquid Release Agent (Electron Microscopy Sciences). Areas of interest were selected based on the DAB labeling and re-embedded in Epon. These re-embedded sections were cut using an ultramicrotome in 70-nm-thick ultrathin sections. These 70-nm sections were examined with a JEOL transmission electron microscope and photographed at primary magnifications of 3000–4000×.