Tbst buffer

The cells were lysed on ice for 30 minutes in RIPA lysis buffer (Meilunbio, China) containing phenylmethanesulfonyl fluoride (0.1 mg/mL). The supernatant was centrifuged to obtain the sample of the protein. The protein was quantified by a BCA kit (Meilunbio, China) with 10 μL of the supernatant taken. 40μg protein sample was loaded to 8% -12% gel electrophoresis SDS-PAGE. The protein was transferred to the polyvinylidene fluoride (PVDF) Membrane (Millipore, USA, ThermoFisher, USA). The PVDF membrane was blocked in a blocking solution by the TBST buffer (Meilunbio, China), which contains 5% skim milk powder (BD, USA) for one hour. The PVDF membrane was incubated with the primary antibody at 4 °C overnight and then incubated with the corresponding secondary antibody for 1 hour at room temperature. After being washed three times with TBST buffer (Meilunbio, China), The PVDF membrane was detected by Western fluorescence assay Beyo ECL Plus (Beyotime, China, Meilunbio, China). The following antibodies were used: Anti-CD44 (1:2000, CST, USA), Anti-EpCAM (1:1000, CST, USA), Anti-MKL-1 (1:1000, CST, USA), Anti-GAPDH (1:1000, Abcam, USA).

The ischemic tissue from the ipsilateral hemisphere of the striatum was dissected and lysed in the protein lysis buffer (RIPA, protease cocktail inhibitor and phosphatase inhibitor) on ice immediately after brain sample collection [31 (link)]. The protein concentration of each protein sample was determined by a BCA kit (Meilunbio, Dalian, China). An aliquot of 30 μg of total protein from each sample was loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and subjected to electrophoresis. The protein samples on the SDS-PAGE gel were transferred to polyvinylidene fluoride membrane (Merck KGaA, Darmstadt, Germany). The membrane was blocked with 5% non-fat milk for 1 hour at room temperature and incubated with the primary antibodies against GFAP (1:4000, AB5804, Millipore, MA), C3d (1:1000, AF2655, R&D system), and β-actin (1:1000, 66009, Invitrogen) overnight at 4°C. After being washed three times in TBST buffer (Meilunbio), the membranes were incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Immunoblots were developed by incubating with solutions from an enhanced chemiluminescence kit (ECL, Pierce). The results of ECL were analyzed using Image J software.