Sequential changes of mE-selectin in vessels, sE-selectin in serum, and mRNA levels for E-selectin in various tissues after induction of limb ischemia in mice. (A) Immunofluorescent staining of mE-selectin (red) and murine EC (BS-1 lectin, green) in muscle after ischemia (scale bar is 200 μm). The expression level of mE-selectin was low in vessels of nonischemic muscle (0 hour). However, it peaked between 6 and 12 hours after ischemia and returned to baseline level by 24 hours. It showed no change at day 7. (B) ELISA analysis showed the changes of sE-selectin concentration in serum after ischemia. sE-selectin increased gradually over 24 hours. It was still high at 7 days (†P < .05; n = 4). ns indicates not significant. (C) Real-time RT-PCR demonstrated the comparison of E-selectin mRNA expression in various tissues after ischemia (n = 4). The level of mRNA for E-selectin in ischemic muscle showed a remarkably high peak around 12 hours after induction of ischemia, whereas nonischemic muscle showed a mild increase at 12 hours. In bone marrow, it was maintained at a high level at day 7. On the other hand, it had been low throughout 7 days in other tissues such as liver and spleen. Error bars in panels B and C represent SD.