Ab252921

Western blot assay was carried out as the previously described methods (Roman et al., 2020 (link)). Primary microglia and mouse microglia BV2 cells with different treatments were collected, and the total proteins were isolated using RIPA buffer (Gibco, United States). Then, the different protein samples were loaded into the lane of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, followed by the transfer into polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, United States). Next, the membranes were placed in 5% skim milk and blocked at RT for 1 h and then incubated with the specific primary antibodies at 4°C overnight. The antibodies were: anti-CD86 (ab243887, 1:1000, Abcam), anti-CD206 (ab252921, 1:1000, Abcam), anti-p-JNK (sc-6254, 1:500, Santa Cruz Biotechnology), anti-JNK(sc-7345, 1:500, Santa Cruz Biotechnology), anti-p-p38 (sc-166182, 1:500, Santa Cruz Biotechnology), anti-p38 (ab170099, 1:2000, Abcam), anti-pERK (sc-7383, 1:500, Santa Cruz Biotechnology), anti-ERK (ab32537, 1:1000, Abcam) and β-actin (ab6276, 1:5000, Abcam). The samples were further incubated with the secondary antibody (ab205718, 1:2000, Abcam) at 37°C for nearly 1 h. Ultimately, the enhanced chemiluminescence reagents (Millipore, Bedford, MA) and ImageJ were applied to analyze protein bands.

Formalin fixed and paraffin embedded liver tissue was cut into 4 μm-thick sections and stained with H+E to evaluate the degree of liver damage. Suzuki’s Scores were calculated to quantify the damage. To identify apoptosis and regeneration of hepatocytes resulting from hepatic IRI, Caspase 3 (1:200, ab109201; Abcam), PCNA (1:200, ab92552; Abcam) and CD206 (1:200, ab252921; Abcam) rabbit antibodies were used for immunohistochemical staining. Image J was used to semi-quantitative analysis. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) reaction was performed using an In Situ Cell Death Detection Kit, TMR red to assess apoptosis. The mean number of TUNEL-positive cells in five different fields (400×) were averaged for quantification.

Here, we used ACTA2, a marker of CAFs, and CD206, a marker of macrophage M2, to discuss the relation between CALD1, CAFs, and macrophage M2. The expression of CALD1 (1:250, Abcam, ab32330), ACTA2 (1:250, Abcam, ab7817), and CD206 (1:4000, Abcam,ab252921) in tumor tissues was detected using the BenchMark GX automatic immunohistochemical staining system (Roche, Switzerland). After deparaffinization, the tissue sections were incubated with primary antibody for 32 min. Biotinylated anti-IgG antibody and horseradish peroxidase were used to show positive expression areas. Hematoxylin was used for counterstaining and Bluing Reagent for post counterstaining.