Characterization of mGluR5 and D1 in Mouse Brain

For immunohistochemical characterization, mice were first deeply anaesthetized with thiopental sodium (150 mg/kg, i.p.) and transcardially perfused with a fixative (4% paraformaldehyde + 15% picric acid in 0.1 M phosphate-buffer (PB), pH 7.2–7.4). Following brain extraction, coronal sections were cut (50 μm) on a Leica VT1000S vibratome (Leica Microsystems, Vienna, Austria) and immunostained against mGluR5 and D1, based on previously described procedures [76 (link)]. A rabbit antibody against mGluR5 (Frontier Institute, Hokkaido, Japan; AB_2571802) and a goat antibody against D1 (Frontier Institute, AB_2571594) were diluted 1:1000 in 2% normal goat serum (NGS), 0.1% Triton X-100 in Tris-buffered saline (TBS; pH 7.4) and sections incubated for 48 h at 6 °C. Sections were then incubated overnight with the respective secondary antibodies (anti-goat Alexa Fluor™488, 1:1000, Jackson ImmunoResearch Europe Ltd.; anti-rabbit Cy3, 1:500, Invitrogen, ThermoFisher Scientific, Vienna, Austria). After three washing steps with TBS, sections were finally mounted onto gelatin-coated slides and coverslipped with Vectashield (Vector Laboratories, Burlingame, US). Immunofluorescent sections were examined using a Zeiss AxioImager M1 microscope or a confocal laser-scanning microscope (SP5, Zeiss, Oberkochen, Germany) for low and high-resolution image acquisition, respectively.