Animals were sacrificed 4–6 weeks post-injection and perfused with 4% PFA. The brains were dissected out and fixed in 4% PFA for 2 hours at room temperature, rinsed with 1X PBS, then placed in 15%–30% sucrose overnight at 4°C. Brains were then rinsed in PBS, embedded in OCT, and frozen at −80°C until sectioning.
35 um sections were cut on a Leica CM1950 cryostat. Free-floating sections were washed in PBS 3× 5 minutes, followed by 1 hour blocking in 5% normal donkey serum (NDS). Primary antibody (goat anti-Fos; Santa Cruz Biotechnology sc-52-G, 1:400) was diluted in 0.1% PBS/Triton X-100 and 5% NDS and incubated overnight (>12 hours) at 4°C. Sections were then washed with PBS three times and incubated with secondary antibody (donkey anti-goat 568; Invitrogen 1:500) overnight at 4°C. Sections were then washed with PBS three times and mounted onto Superfrost Plus slides, dried >15 minutes room temperature, and coverslipped in 50% glycerol containing DAPI (1 ug/mL). Images were acquired using a confocal microscope (Zeiss LSM 880).
35 um sections were cut on a Leica CM1950 cryostat. Free-floating sections were washed in PBS 3× 5 minutes, followed by 1 hour blocking in 5% normal donkey serum (NDS). Primary antibody (goat anti-Fos; Santa Cruz Biotechnology sc-52-G, 1:400) was diluted in 0.1% PBS/Triton X-100 and 5% NDS and incubated overnight (>12 hours) at 4°C. Sections were then washed with PBS three times and incubated with secondary antibody (donkey anti-goat 568; Invitrogen 1:500) overnight at 4°C. Sections were then washed with PBS three times and mounted onto Superfrost Plus slides, dried >15 minutes room temperature, and coverslipped in 50% glycerol containing DAPI (1 ug/mL). Images were acquired using a confocal microscope (Zeiss LSM 880).