Horse anti mouse secondary antibody

Coronal sections (40 µm) were cut using a sliding microtome (Model SM2000R; Leica Microsystems, Bannockburn, IL, USA). Three sections each 1 mm apart were selected for processing (anterior to bregma, in mm: 2.6, 1.6, and 0.6). M1 was analyzed in all three slices and the striatum was examined in the two posterior sections. Prior to processing and between incubations, all slices were triple-washed with phosphate-buffered saline. Sections were first immersed in 0.3% H₂O₂ for 30 min. Subsequently, slices were placed in blocking buffer (phosphate-buffered saline with 1% bovine serum albumin, 1% normal goat serum and 0.4% Triton X-100 [Sigma-Aldrich]) for 120 min at room temperature. Sections were then incubated in a mouse anti-TH primary antibody (1:500 dilution in blocking buffer; Millipore, Billerica, MA, USA) at 4° C overnight. Next, slices were bathed in a horse anti-mouse secondary antibody (1:200 dilution in blocking buffer; Vector Laboratories, Burlingame, CA, USA) at 4° C for 120 min. Sections were then incubated for 1 h in a horse radish peroxidase-conjugated avidin-biotin mixture (VectaStain Elite ABC Kit, Vector Laboratories). For the chromagen step, SigmaFast kits (Sigma-Aldrich) containing 3,3’-diaminobenzidine and H₂O₂ were prepared according to manufacturer’s instructions. After staining, slices were mounted on glass slides, dehydrated, defatted and cover slipped.