Immunohistochemical Analysis of Brain Regions

Three brain sections per animal were selected to contain the ventral posteromedial (VPM)/ventral posterolateral (VPL) nuclei of the thalamus and the somatosensory barrel field (S1BF) cortex. Immunohistochemistry was performed as previously described^{66 (link)}. Briefly, free floating brain sections were incubated in 1% hydrogen peroxide in Tris-buffered saline (TBS) for 20 min to block endogenous peroxidase activity followed by 3 washes in TBS. Sections were then blocked in 15% goat serum-containing TBS-T (TBS with 0.3% Triton X-100) for 30 min followed by primary antibody solution overnight at 4 °C. Primary antibodies included: anti-CD68 (BioRad AbD Serotec, MCA1957; 1:2,000), anti-GFAP (Dako, Z0334; 1:8,000), and anti-ATP synthase C (Abcam, ab181243, 1:1,000) diluted in TBS-T+ 10% goat serum. Secondary antibodies included: biotinylated anti-rat and anti-rabbit IgGs (Vector Labs, BA-9400, BA-1000; 1:2000) diluted in TBS-T+ 10% goat serum. An ABC amplification kit (Vector Labs) was used for 2 h followed by 0.05% DAB solution until visible reaction had occurred.