5 lox

The cortex was dissected from control and embolic stroke rats according to Spijker [27 ]. Cortical tissues were lysed by RIPA buffer (Cell Signaling Technologies, USA) supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany). Total protein was determined by the Bradford protein assay (Bio-Rad, CA, USA). Proteins were separated by 10% SDS/PAGE, transferred onto a PVDF membrane (Amersham Biosciences, UK) and then blocked with 10% non-fat milk. The membrane was then incubated with antibodies against 5-LOX (1:1000, BD Bioscience), GFAP (1:5000, Chemicon, Merck, USA, Cat. No. MAB3402), ED-1 (1:1000, Chemicon, Merck, Germany, Cat. No. MAB1435), OX-42 (1:1000, Chemicon, Merck, Germany, Cat. No. CBL1512), or β-actin (Cell Signaling Technologies, USA, Cat. No. mAb#4970) at 4 °C overnight, then washed and incubated in HRP-conjugated anti-rabbit or mouse IgG at room temperature for 1 h. Visualization was carried out using Luminata Forte or Crescendo Western HRP substrate (Millipore Corporation, USA) and chemiluminescence signals were detected using UVIchemi (UVItec, UK).