Avidin biotin peroxidase

At 24 h after MCAO or post-stroke infection, mice were anesthetized and transcardially perfused with 20 ml cold PBS and 20 ml of 4% paraformaldehyde in 0.1 M PBS. The brain, lung, liver, and spleen were removed, post-xed, and embedded in para n. The tissue blocks were cut into 5-mm sections that were depara nized and stained with HE according to standard protocols. CD8 + T cells and NK cells in the brain and spleen were identi ed by immuno uorescence analysis and immunohistochemistry, as previously described. For the latter, brain and spleen tissue sections were incubated overnight at 4°C with primary antibodies against CD8 (ab25117) and natural cytotoxicity receptor (NCR) (ab199128),Iba1(ab5076),CD68(ab125212)(Abcam,Cambridge,MA,USA) respectively followed by processing with avidin-biotin-peroxidase (BosterBio, Wuhan, China). The sections were stained with diaminobenzidine, and nuclei were counterstained with hematoxylin.
For immuno uorescence, the specimens were rst treated with anti-CD8 or -NCR antibody, followed by Alexa Fluor 594-conjugated secondary antibody (A0453; Beyotime Institute of Biotechnology, Shanghai, China). Immuno uorescence images were acquired with a confocal laser scanning microscope (TCS SP2; Leica Microsystems, Wetzlar, Germany).