Immunofluorescence Assay for VGlut2 Expression

The immunofluorescence technique was performed as previously described (Gerig and Celio, 2007 (link); Mészár et al., 2012 (link)). In short, CTR, HET, and KO mice (n = 3/genotype) were perfused with 0.9% NaCl, followed by 4% paraformaldehyde, diluted in 0.1M phosphate buffered saline, pH 7.3. The brains were dissected and immersed in 30% sucrose for one night, and then frozen on dry ice. Eighty micrometers of thin cryomobile (Reichert Jung) sections were prepared and incubated in parallel in 24-well plates with the primary antiserum against VGlut2 (Synaptic Systems, Göttingen, Germany), diluted 1:10,000 to 1:20,000. The efficacy and specificity of this antiserum against VGlut2 was established by immunoblotting experiments (see section Western Blot of Brain Extracts). Incubation with the biotinylated secondary antibody was followed by exposure to streptavidine-Cy3 (Alexa 550). The sections were mounted on glass slides for histological inspection in either a Leica 6,000 epifluorescence microscope (equipped with a Hamamatsu C4742-95 camera) or a digital slide-scanner (Nanozoomer, Hamamatsu).