Donkey anti mouse conjugated to alexa fluor 488

Slides were brought to room temperature and then the perimeter of each slide was traced with a hydrophobic pen and soaked 3× for 10 minutes in phosphate buffered saline (PBS; pH 7.2), followed by a 1 hour soak in a blocking solution made of PBS with 0.3% Triton X-100 and 10% normal donkey serum (PBS-DS). Following the blocking procedure, slides were incubated for 16–17 hours at room temperature in PBS-DS containing rabbit anti-cFos (1:2000, Santa Cruz Biotechnology lot# C2510) and mouse anti-Hu, a specific marker for neuronal somata (1:2000, Molecular Probes). After incubation, slides were briefly dipped (to remove the majority of the antibody solution) and then rinsed in PBS + 0.5% normal donkey serum 5× for 10 minutes. The slides were then incubated for 2 hours at room temperature with PBS-DS containing donkey anti-rabbit conjugated to Alexa Fluor 568 (1:200, Life Technologies) for cFos and donkey anti-mouse conjugated to Alexa Fluor 488 (1:200, Life Technologies) for Hu. Finally, slides were dipped and rinsed 4× for 10 minutes in PBS before being cover slipped using ProLong Gold with DAPI and then allowed to cure for 48–72 hours in a dark room. Once dry, the slides were sealed with nail polished and stored at 4°C.