Visualizing Neurons Expressing ChR2 via c-Fos

To assess activation of neurons expressing ChR2, immunofluorescent labeling of the intermediate-early gene c-Fos was carried out. Brain tissue was rinsed in 50 mM KPBS (pH 7.4) and incubated in blocking solution (KPBS, 0.1% BSA, and 0.2% TritonX-100) for 1 h at room temperature. Brain slices were then transferred into rabbit polyclonal anti-c-Fos (sc-52) primary antibody (1:1,000; Santa Cruz Biotechnology, Dallas, TX) overnight at 4°C (Jones et al. 2011 (link)). The next day, brain tissue was rinsed in KPBS and incubated in Cy3-conjugated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch) for 30 min, followed by a final rinse. Sections were then mounted onto slides and cover slipped. To quantify specificity of the CaMKIIα promoter, immunofluorescent labeling of the GABAergic marker glutamic acid decarboxylase 67 (GAD67) was carried as described above using mouse monoclonal anti-GAD67 primary antibody (1:1,000; Millipore) and Cy3-conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch).