Photooxidation-mediated GFP Neuron Labeling for EM

Photooxidation-mediated specific labeling of GFP-expressing neurons for EM was done as described by Horstmann et al. (2013 (link)). Briefly, perfusion-fixed (4% PFA) brain slices from animals injected with AAV-synaptophysin-2-pHluorin were cut at 200 μm thickness. The slice of interest was incubated in an oxygenated Tris-HCl buffer overnight. The following day, the pHluorin was excited under a light microscope in a 1 mg/mL 3,3′-Diaminobenzidine (DAB) supplemented Tris-HCl buffer. The subsequently excised region of interest with the DAB-precipitate was embedded in epoxy resin. For EM the tissue block was serially sectioned at a thicknes of 38 nm using an Ultracut S ultramicrotome (Leica, Germany) equipped with a diamond knife angled at 35° (Diatome, Biel, Germany). The 109 sections were collected onto hydrophilized silicon wafer strips and post stained with Reynolds lead citrate.