Sm2010r microtome

Deeply anesthetized mice (with tribromoethanol, 250 mg/kg, i.p.) were transcardially perfused using PBS followed by 4% PFA. After post-fixation for 24 h, the brains were cryopreserved and cryosectioned at 30 μm with a Leica SM2010R microtome. For immunofluorescence, the brain slices were washed 3X in PBS, permeabilized in 0.5% Triton X-100-PBS solution for 0.5 hour, and then blocked in blocking buffer (10% normal donkey serum/NDS, 0.1% Triton X-100-PBS solution) for 1 hour. Following the blocking procedure, slices were incubated with primary antibodies diluted in the blocking buffer (Table 4) overnight at 4°C with agitation. The next day, slices were washed 3X in PBS and then incubated with specific fluorescent dye conjugated secondary antibodies diluted in PBS (Table 4) for 2 hours. Finally, they were mounted with Vectashield antifade mounting medium (Vector Laboratories: H-1000–10, Newark, CA, USA) on glass slides. Fluorescent images were acquired via an Olympus FV3000 confocal microscope. Images were then processed with ImageJ Fiji software. Pseudo-coloring was applied in certain images to improve visibility.

Mice were perfused intracardially with 4% paraformaldehyde in 0.01 M phosphate-buffered saline and the brains removed. The brains were incubated overnight at 4°C in 4% paraformaldehyde and then incubated in 30% sucrose for cryoprotection. Next, the brains were frozen on dry ice and cut into 40 μm coronal sections on a Leica SM2010R microtome, and the sections were placed in cryoprotectant (0.01 M PBS, formula) before storage at −20°C. The first section containing striatum was identified and placed in the first well of a 24-well plate, then the next nine serial sections were placed in the next nine wells. Next, the 11th section is placed in the first well and the next nine wells placed in the subsequent wells, such that the first 10 wells contain four sections each, where each section in the well is separated by 400 μm. A total of 14 wild-type and 14 BACHD mice were used for the experiments performed: five wild-type (3 M, 2 F) and 5 BACHD (2 M, 3 F) mice were analyzed for parvalbumin expression, three wild-type (2 M, 1 F) and three BACHD (2 M, 1 F) mice were analyzed for somatostatin expression, three wild-type (2 M, 1 F) and three BACHD (1 M, 2 F) were analyzed for calretinin expression, and three wild-type (1 M, 2 F) and three BACHD (1 M, 2 F) were analyzed for ChAT expression.