Alexa fluor 488 green conjugated goat anti rabbit igg

Cells seeded in 24-well plates were washed twice with ice-cold PBS, fixed with 100% methanol for 7 min at –20°C, and permeated with fresh 4% paraformaldehyde for 20 min at room temperature. These cells and the coronal cryostat sections through the hippocampi were blocked with blocking buffer (10% goat serum in PBS containing 0.3% Triton X-100 and 0.03% NaN₃) overnight at 4°C. The cells and sections were then incubated with the primary antibody diluted in blocking buffer at 4°C for 24 h, followed by incubation with the secondary antibody overnight at 4°C. After further washing with PBS, the cells and sections were stained with Hoechst (1∶1,000; Pierce, Rockford, IL, USA) for 0.5 h at room temperature, and then viewed under a fluorescence microscope (Leica DMIRB, Germany). Primary antibodies were as follows: mouse monoclonal anti-choline acetyltransferase (ChAT) (1∶500, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-Brn-4 (1∶200, Santa Cruz, CA, USA), mouse monoclonal anti-microtubule-associated protein (MAP)-2 (1∶1,000; Millipore, Billerica, MA, USA) and rabbit polyclonal anti-Brn4 (1∶500; Santa Cruz, CA, USA). Secondary antibodies were: Alexa Fluor 568-conjugated (red) goat anti-mouse IgG (1∶500; Invitrogen, Carlsbad, CA, USA), and Alexa Fluor 488-conjugated (green) goat anti-rabbit IgG (1∶200; Invitrogen, Carlsbad, CA, USA).