Co-immunoprecipitation (IP)

Co-IP was performed to analyze the ICAM-1, LFA-1, and Mac-1 protein-protein interactions using Pierce Classic Magnetic IP/Co-IP kit (catalog #88804). Briefly, brain tissue (50 mg) from WT and ICAM-1^−/− mice with and without FPI were harvested and lysed with 500 μl of IP Lysis/Wash buffer and following lysis and centrifugation, the supernatant was transferred to a new tube for protein concentration determination. For this experiment, Pierce Protein A/G Magnetic Beads were used for IP according to the manufacturer’s protocol. Briefly, 500 μg of total protein extract per sample was subjected to IP assay using 10 μg of anti-ICAM-1 antibody and incubated overnight at 4°C. After the formation of the immune complex, A/G Magnetic Beads were added and incubated for 1 h. After washing the IP complexes, the samples were suspended in 200 μl of 2xSDS sample buffer. Twenty microliters of aliquots were then subjected to Western blotting using ICAM-1, LFA-1, and Mac-1 antibodies.