Enzyme‐linked immunosorbent assay (ELISA) for determining cytokine profiles

Brain tissue was taken from ischaemic regions. Simultaneous quantitation of 12 cytokines (IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐6, IL‐10, IL‐12, IL‐17A, interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α, granulocyte colony‐stimulating factor (G‐CSF) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) was accomplished using a multi‐analyte ELISArray Kit (Qiagen, Düsseldorf, Germany). Procedures followed the manufacturer's protocol. Each sample was analysed in triplicate from individual mouse brains (six mice per group), and results are expressed as mean optical density (OD) ± standard error of the mean (s.e.m.).