Application suite advanced fluorescence 3 0 0 build 8134

Ca_v1.2 fluorescent immunohistochemistry was performed to confirm elimination of Ca_v1.2. Fluorescent immunohistochemistry was also used to confirm injection placement. Mice were transcardially perfused with 4% PFA, and brains were dissected and postfixed overnight in 4% PFA followed by cryoprotection in 30% sucrose at 4°C for at least 72 h. Forty-micrometer-thick sections spanning the hippocampus were obtained using a sliding microtome and incubated in anti-chicken GFP (1:10,000, Aves Labs) and anti-rabbit glial fibrillary acidic protein (1:1000, Invitrogen) primary antibody overnight at 4°C. Sections were rinsed in 0.1 m phosphate-buffer (PB) and incubated with donkey AlexaFluor 488 (1:300) and AlexaFluor 568 (1:300) antibody for 1 h at room temperature. Doublecortin fluorescent immunohistochemistry was performed to analyze cells in the dentate gyrus that had recently committed to neuronal fate. Sections were incubated in anti-guinea pig doublecortin (1:5000, Millipore) primary antibody overnight at 4°C. Sections were rinsed in 0.1 m PB and incubated with donkey AlexaFluor 594 (1:400) antibody for 1 h at room temperature. Sections were imaged using an epifluorescent microscope (Leica DM550B with Leica Application Suite Advanced Fluorescence 3.0.0 build 8134 software, Leica Microsystems).