Brains and spinal cords were isolated at day 16 after EAE induction from perfused mice and cut into 2 mm thin sections. Tissues were washed three times in 1 ml PBS and subsequently fixed and permeabilized in 4% PFA/0.1% Triton-X-100 in PBS on a shaker (1 h, 800 rpm, 4 °C). Afterward, sections were incubated with blocking buffer (1% BSA/0.1% Triton-X-200 in PBS) on a shaker (1 h, 800 rpm, 4 °C). Samples were washed three times in PBS and primary antibodies against P2YR12 (Biolegend, S16007D) diluted in blocking buffer were added for 48 h. Samples were washed four times in PBS for 30 min on a shaker and incubated with the appropriate secondary antibody on a shaker (48 h, 800 rpm, 4 °C). Confocal imaging of brain regions was performed on a Zeiss LSM880 NLO Two-Photon microscope, equipped with a 680–1300 nm tunable and a fixed 1040 nm two-photon laser from Newport SpectraPhysics. Two-photon images of brain regions were acquired with a 20x W-Plan Apochromatic objective water dipping lens. The fluorophores AF633 (for P2YR12) and eGFP were excited at 940 nm, and specific emission was detected at 500–550 nm and 640–710 nm.
