Protein Expression Analysis of Ischemic Penumbra

Fifty milligrams of ischemic penumbra tissues (n = 6) was used for Western blot analysis as previously described [15 (link)]. The primary antibodies include the following: mouse anti-VE-cadherin (1:1000, Cell Signaling Technology), rabbit anti-AQP4 (1:1000, Proteintech, USA), rabbit anti-Claudin-1 (1:1000, Abcam, USA), mouse anti-GFAP (1:1000, Cell Signaling Technology), rabbit anti-Iba1 antibody (1:500, Wako), goat anti-C3d antibody (1:1000, RD Systems), rabbit anti-S100A10 (1:1000; Invitrogen), rabbit anti-TLR4 antibody (1:1000, Cell Signaling Technology), mouse anti-phospho-NF-κB p65 (Ser536) antibody (1:1000, Abcam), mouse anti-NF-κB p65 antibody (1:1000, Abcam), mouse anti-GAPDH (1:1000, Cell Signaling Technology). The membranes were then incubated with the corresponding HRP-conjugated secondary antibody for 2 h. Protein bands were visualized using the LI-COR Odyssey System (LI-COR Biotechnology, USA).