Bs 0698r

Total protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) from heart tissue and HL-1 cells, and the concentration of the protein was detected by the BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Next, about 20 µg protein was subjected to 12% SDS-PAGE and then transferred on PVDF membrane (Sangon, Shanghai, China). The PVDF membranes were washed with water and stained with Ponceau S to confirm transfer, after that blocked with TBST solution contained 5% skimmed milk at 4 °C overnight. Later, PVDF membranes were incubated with primary antibodies at 4 °C overnight followed by incubated with secondary antibody Goat Anti-Mouse IgG H&L(HRP) (1:10000, ab205719, Abcam, Cambridge, UK) at 25 °C for 2 h. The protein bands were imaged by chemiluminescence apparatus (Shanghai Qinxiang, China) and quantified by Image J software. Primary antibodies including: Wnt1 (1:1000, sc514531, Santa Cruz), β-catenin (1:1000, 8480T, Cell Signaling Technology), IL-10 (1:1000, bs-0698R, Bioss), NOS2 (1:1000, sc7271, Santa Cruz), CD63 (ab68418, Abcam, Cambridge, UK), ACSL4 (1:10000, ab155282, Abcam, Cambridge, UK), TFRC (1:1000, ab214039, Abcam, Cambridge, UK), GAPDH (1:20000, 60004-1-Ig, Proteintech).

Brain and colon tissues were paraffin-embedded, processed into 5 μm thick slices, and dewaxed in an organic solvent concentration gradient. Tissue sections were placed in a microwave oven for antigen retrieval, immersed in a 3% hydrogen peroxide solution at room temperature, and protected from light for 25 min to block endogenous peroxidase. After the slices were dried, primary antibodies against IL-6 (bs-0782R, Bioss, Beijing, China), IL-10 (bs-0698R, Bioss, Beijing, China), TNF-α (bs-10802R, Bioss, Beijing, China), ZO-1 (ab96587, Abcam, Cambridge, UK), and occludin (ab216327, Abcam, Cambridge, UK) were diluted in 3% normal goat serum and applied to the tissue. The sections were placed in a wet box and incubated overnight at 4 °C. After washing, an HRP-conjugated secondary antibody (ZSGB Biotech, Beijing, China) was applied to the tissue. The nuclei were stained with hematoxylin, and the tissue sections were sealed with neutral gum. Images were captured using an Olympus BX61 light microscope. The digital images were analyzed using Image Pro Plus (version 6.0), as previously described [21 (link),22 (link)]. The gray unit was switched to the optical density (OD) unit in the software, and the integrated OD analysis was performed with immunohistochemically stained regions.