Hrp conjugated anti rabbit or anti mouse igg

Ischemic cortex tissue samples were extracted at 3 dpi and homogenized in RIPA lysis buffer. After SDS-PAGE and protein transfer, membranes were incubated with primary antibodies including rabbit anti-IL-17 (1: 1 000, Abcam, Cat# ab79056), mouse anti-Wnt2 (1: 5 000, Abcam, Cat#66656-1-lg), rabbit anti-p-PI3K p85 ((Tyr458)/p55 (Tyr199), 1:1 000, Cell signaling, Cat#17366), rabbit anti-PI3K p-85(1:1 000, Cell signaling, Cat#4257), rabbit anti-p-Akt (Ser473, 1:1 000, Cell signaling, Cat#4060), rabbit anti-Akt (1:1 000, Cell signaling, Cat#4685), rabbit anti-Caspase 3 (1:1 000, Cell signaling, Cat#14220), rabbit anti-β-catenin (1:1 000, Cell signaling, Cat#25362), rabbit anti-p-GSK-3β (Ser9, 1:1 000, Cell signaling, Cat#5558), rabbit anti-GSK-3β (1:1 000, Cell signaling, Cat#12456), and rabbit anti-β-actin (1:3 000, Cell signaling, Cat#5125) overnight at 4 °C, followed by incubation with HRP-conjugated anti-rabbit or anti-mouse IgG (1: 5 000, Proteintech, USA) for 3 h at room temperature. Bands were visualized with an ECL kit (Thermo).

Western blotting was used to detect the expression of proteins involved in apoptosis, angiogenesis and the MEK/ERK signalling pathway at 24 h after administration. Total protein was extracted using protein extraction kit, according to the manufacturer’s instructions and the protein concentration were measured with BCA method. Protein samples (40 μg) were subjected to 10% SDS-polyacrylamide gels electrophoresis (SDS-PAGE) to separate. Then transferred to poly-vinylidnene fluoride (PVDF, Millipore) membranes and blocked in PBST solution with 5% non-fat milk. The membranes were incubated in primary antibodies against brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), Bax, Bcl-2, VEGF, FGF2, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2 (1:2000 dilution, Proteintech, USA) at 4 °C overnight. Secondary antibodies: HRP-conjugated anti-mouse or anti-rabbit IgG (1:5000, Proteintech, USA) were incubated 1 h at 37 °C. Finally, the enhanced chemiluminescent reagent (Thermo Fisher, USA) was added in the membranes and band intensity signals were observed. GAPDH was used as an internal reference, and the optical densities of protein bands were analysed by Image J software to represent the relative expression of target protein.