Ab213612

The cells or exosomes were lysed with RIPA lysate (Sigma) to obtain proteins. The BCA Assay kit (Solarbio, Beijing, China) was used to determine the protein concentration. An equal amount of protein was added onto 10% SDS-PAGE gels, and the protein was then transferred onto polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The blots were then incubated with primary antibodies, Anti-PI3Kγ antibody (1 : 1,000, ab32089, Abcam), Anti-CD163 antibody (1 : 1,000, ab213612, Abcam), Anti-CD206 antibody (1 : 1,000, sc-58986, Santa Cruz Biotechnology), Anti-PTEN antibody (1 : 1,000, ab267787, Abcam), Anti-AKT antibody (1 : 1,000, ab213612, Abcam), Anti-pAKT antibody (1 : 1,000, ab38449, Abcam), Anti-Vimentin antibody (1 : 1,000, ab20346, Abcam), Anti-α-SMA antibody (1 : 1,000, ab108424, Abcam), Anti-IL-10 antibody (0.5 µg/ml, ab134742, Abcam), Anti-CD63 antibody (1 µg/ml, ab38418, Abcam), Anti-CD61 antibody (1 : 1,000, ab119992, Abcam), Anti-HSP70 antibody (1 : 1,000, ab2787, Abcam), and anti-Actin (1 µg/ml, ab8286, Abcam) overnight at 4°C. The horseradish peroxidase-conjugated secondary antibody was then added to incubate the blots at room temperature for 2 h. At last, the blots were visualized with the Novex™ chemiluminescent substrate reagent kit (Waltham, MA, USA).

Three-μm-thick slices stained with H&E or immunohistochemical (IHC) were obtained from formalin-fixed paraffin-embedded (FFPE) EEAs tissues, which were the same wax blocks as the previous nanostring analysis. IHC staining of FFPE slides was performed using monoclonal mAbs against BIRC5(ab76424), CD68(ab213363) and CD163(ab213612) (Abcam, Cambridge, UK), with 1:500 working dilution. Paraffin sections are first dewaxed in steps from xylene to different concentrations of alcohol, and finally placed in tap water for washing. The slices were treated with heated citric acid repair fluid for antigen repair. After incubation with 3% H2O2 for 10 min, endogenous peroxidase was removed. Goat serum was used for blocking for 30 min. The first antibody was incubated overnight at 4 degrees. The next day, the second antibody labeled with horseradish peroxidase was incubated for 30 min. In the middle of each step, 1 x PBS should be used for cleaning at 5 min * 3 times. DAB was used for IHC staining observed under the microscope.
Scoring for BIRC5, CD68, and CD163 was evaluated by percentage of cells stained in tumor and stromal tissue compartments by a pathologist.