Rabbit anti d2r

Mice were euthanized by CO₂ asphyxiation followed by rapid brain removal. Brains were fixed in 4% paraformaldehyde (Sigma Aldrich) for 24 h and then immersed in 30% (w/v) sucrose in 0.1M PBS at 4 °C for 48 h until the tissue sunk. Coronal free-floating sections (40 μm) were cut by a cryostat and incubated in 0.5% Triton-PBS for 15 min and then in 5% (w/v) bovine serum albumin (BSA)-PBS for 1 h at room temperature. Sections were then incubated with rabbit anti-c-Fos primary antibody (1:100, Cat No.: sc-52, Santa Cruz Biotechnology), rabbit anti-D2R (1:100, Cat No.: sc-9113, Santa Cruz Biotechnology), or mouse anti-NeuN (1:1000, Cat No.: Ab10424, Abcam) at 4 °C overnight. After washing with PBS, slices were incubated with secondary antibody (1:500, Alexa 594-conjugated goat anti-rabbit or Alexa 405-conjugated goat anti-mouse; Abcam) for 3 h at room temperature. Images from the brain region of interest (NAc) were obtained on a LSM 510 confocal laser scanning microscope (Carl Zeiss) with a 20X objective. Quantification of c-Fos positive cells in the NAc per mm² was carried out using 3–5 coronal sections per mouse, using a minimum of 3 mice per group. The total number of c-Fos positive cells present in the NAc from all sections of each mouse was averaged.