For tissue, 50–100mg sections of both infarct and remote zone were homogenized in lysis buffer (20mM Tris, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM β-glycerol, 2.5 mM Na pyrophosphate, and 1% Triton x-100) in varying volumes according to weight of sections supplemented with phosphatase and protease inhibitor cocktails (Roche). Protein concentration was measured using BioRad BCA assay (Bio-Rad, Hercules, CA USA) and samples were prepared in 2X SDS at 15μg/15μl.
For cells, media was removed and immediately used for zymography or stored at −80°C until usage with no addition of protease or phosphatase inhibitors. Cells were washed with ice cold PBS, then lysed with lysis buffer (20mM Tris, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM β-glycerol, 2.5 mM Na pyrophosphate, and 1% Triton x-100) supplemented with phosphatase and protease inhibitor cocktails (Roche). The cells were then frozen at −80 for 1 hours, removed and allowed to thaw on ice and then insoluble material pelleted by centrifugation in a tabletop microcentrifuge at 4°C. Protein concentration was measured using BCA assay (Bio-Rad, Hercules, CA USA) and samples prepared with 4X SDS at 15ug/15ul.
For cells, media was removed and immediately used for zymography or stored at −80°C until usage with no addition of protease or phosphatase inhibitors. Cells were washed with ice cold PBS, then lysed with lysis buffer (20mM Tris, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM β-glycerol, 2.5 mM Na pyrophosphate, and 1% Triton x-100) supplemented with phosphatase and protease inhibitor cocktails (Roche). The cells were then frozen at −80 for 1 hours, removed and allowed to thaw on ice and then insoluble material pelleted by centrifugation in a tabletop microcentrifuge at 4°C. Protein concentration was measured using BCA assay (Bio-Rad, Hercules, CA USA) and samples prepared with 4X SDS at 15ug/15ul.