Vcam 1

All samples were repaired in a microwave and incubated in 0.3% hydrogen peroxide before using primary [vascular cell adhesion molecule-1 (VCAM-1), BA3840, Boster, China] and secondary antibodies (Zhongshan Golden Bridge Biotechnology Company, China). Positive reactions were visualized by incubating the slides with diaminobenzidine as a substrate in the cytomembrane. Images were photographed with SPOT V3.0II software and the average optical density was measured and analyzed by Image-Pro Plus 6.0.

Paraffin sections (2 μm in thickness) were used for all staining. Briefly, following blocking endogenous peroxidase with PBS containing 3% hydrogen peroxide for 5 min, sections were stained with primary antibodies for neutrophils (myeloperoxidase, MPO, Cat#: bs-4943R, 1:200, Bioss, Boston, MA, USA), macrophages (F4/80, Cat#: 28463-1-AP, 1:1000, Proteintech, Wuhan, Hubei, China), intercellular adhesion molecules-1 (ICAM-1, Cat#: 10020-1-AP, 1:1000, Proteintech), vascular adhesion molecule-1 (VCAM-1, Cat#: BM4289, 1:1000, BOSTER, Wuhan, Hubei, China), and P-gp (Cat#: 13978S, 1:1000, Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. Then, sections were incubated with Immunohistochemical staining kit (DAKO, Carpinteria, CA, USA) for 1 h at room temperature. All stained sections were counterstained with Giles’ hematoxylin and imaged on a fluorescent inverted microscope (Ts2R, Nikon). To quantify P-gp expression levels, neutrophil and macrophages, randomly selected fields from the ischemic cortex were analyzed by using ImageJ software.