Iba1 or glial fibrillary acidic protein (GFAP) immunohistochemistry

To evaluate the effect of S1P₃ activity on microglia or astrocyte activation, Iba1 or GFAP immunohistochemistry was performed 1 or 3 days after tMCAO. Brain sections were oxidized with 1% H₂O₂ in PBS for 15 min and blocked with 1% fetal bovine serum (FBS) in 0.3% Triton-X100 in PBS for 1 h to block non-specific protein binding. Then, the brain sections were incubated with primary antibody against Iba1 (1:500, Wako) or GFAP (1: 500, Invitrogen) overnight at 4 °C followed by anti-rabbit secondary antibody (1:200). Sections were exposed to avidin and biotinylated horse-radish peroxidase macromolecular complex (ABC) kit (1:100, Vector Labs) and visualized with 3, 3′-diaminobenzidine tetrahydrochloride (DAB) exposure (0.02% DAB and 0.01% H₂O₂ in 0.05 M TRIS solution), dehydrated with ethanol, cleared in xylene, and mounted using mounting media.