To measure the in vitro affinity of APN and SAPN to CD147, bEnd.3 cells were seeded on the coverslips in a 24-well plate. When the cells formed confluent monolayers, the previous medium was removed, and they were washed thrice with PBS and then incubated with serum-free medium containing Rhodamine-labeled APN or SAPN (10 nM) for 1 h. After that, cells were washed twice with PBS, fixed with 2% PFA in PBS solution, immunostained for CD147 (1:200, Abcam), and visualized by fluorescence microscopy.
To test the in vivo affinity of APN and SAPN to CD147, both normal mice and tMCAO mice were injected with Rhodamine-labeled APN or SAPN solution (corresponding to AP9 of 2.5 mg/kg body weight, and rhodamine of 15 μmol/kg body weight) at 1 h after tMCAO onset. After transcardial perfusion with cold PBS (pH 7.4) under deep anesthesia at 4 h after induction of tMCAO, brains were immediately removed, fixed in 4% paraformaldehyde overnight, rinsed several times in PBS, then stored in 30% sucrose at 4 °C until the brains sank. Brains were embedded in optimal cutting temperature compound (OCT compound) prior to frozen sectioning to 6 μm thick coronal sections. Sections were further immunostained for CD147 (1:200; Abcam) as we described previously.12 (link)
To test the in vivo affinity of APN and SAPN to CD147, both normal mice and tMCAO mice were injected with Rhodamine-labeled APN or SAPN solution (corresponding to AP9 of 2.5 mg/kg body weight, and rhodamine of 15 μmol/kg body weight) at 1 h after tMCAO onset. After transcardial perfusion with cold PBS (pH 7.4) under deep anesthesia at 4 h after induction of tMCAO, brains were immediately removed, fixed in 4% paraformaldehyde overnight, rinsed several times in PBS, then stored in 30% sucrose at 4 °C until the brains sank. Brains were embedded in optimal cutting temperature compound (OCT compound) prior to frozen sectioning to 6 μm thick coronal sections. Sections were further immunostained for CD147 (1:200; Abcam) as we described previously.12 (link)
