Ab183322

Samples were fixed in 10% (v/v) formaldehyde in PBS, embedded in paraffin and cut into 4-µm sections and used for IHC staining with antibodies. To enhance antigen exposure, the slides were treated with 1X EDTA at 98°C for 10 min for antigen retrieval. The slides were incubated with endogenous peroxidase blocking solution to inhibit endogenous peroxidase and were then incubated with the primary antibody (CD14, ab183322; Abcam, dilution 1:100; MPO, ab208670; Abcam, dilution 1:1,000; NCF2, Abs135832, Absin, dilution 1:100; SOD2, ab246860; Abcam, dilution 1:1,000; PARP1, ab194586; Abcam, dilution 1:50; MUT, ab240091; Abcam, dilution 1:200; ACADM, ab239914; Abcam, dilution 1:500; PCK1, ab248573; Abcam, dilution 1:200) at room temperature for 60 min. After rinsing with Tris-buffered saline, the slides were incubated for 45 min with biotin-conjugated secondary antibody (catalog no. SA00004-2; Proteintech Wuhan Sanying, dilution 1:600), washed, and then incubated with enzyme conjugate HRP-streptavidin. Freshly prepared DAB (Zymed; Thermo Fisher Scientific, Inc.) was used as a substrate to detect HRP. Finally, slides were counterstained with hematoxylin and mounted with aqueous mounting media. Studies on human tissue samples were conducted with approval from the Ethics Committee of the Sir Run Run Shaw Hospital, Zhejiang University.

Paraffin-embedded tissues of 24 cases from CGGA cohort were collected for immunohistochemical staining (Supplementary Table S2). In brief, the sections were incubated with antibodies overnight at 4 °C, and then with secondary antibodies at room temperature for 1 h. After staining with DAB (MXB, Fuzhou, Fujian, PR China) for 1min, sections were counterstained with hematoxylin (Solarbio, Tongzhou, Beijing, PR China). The used antibodies included anti-CD163 (16646-1-AP, 1:300, RRID: AB_2756528, Proteintech, Wuhan, Hubei, PR China) and anti-CD206 (18704-1-AP, 1:300, RRID: AB_10597232, Proteintech) for M2 macrophages, anti-CD3 (ab16669, 1:300, RRID: AB_443425, Abcam, Burlingame, CA, USA) for lymphocytes, anti-CD14 (ab183322, 1:300, RRID: AB_2909463, Abcam) for monocytes, anti-TIGIT (99567, 1:300, RRID: AB_2922806, Cell Signaling, Danvers, Massachusetts, USA), anti-HAVCR2 (60355-1-1g, 1:300, RRID: AB_2881464, Proteintech), anti-MET (8198, 1:300, RRID: AB_10858224, Cell Signaling), anti-EGFR (4267, 1:300, RRID: AB_2246311, Cell Signaling), anti-NOTCH1 (20687-1-AP, 1:300, RRID: AB_10700012, Proteintech). All antibodies were validated by the commercial vendor. Six fields of each section were selected for quantitative analysis, and stained cells were counted by two investigators independently.

Total protein of cells was lysed with RIPA lysis buffer (Abmole, USA) and then centrifuged at 12,000 rpm at 4°C for 15 mins. SDS-PAGE, 10%, gel was used to separate the protein samples, and the proteins were transferred to the PVDF membrane (Bio-Rad, USA), which was blocked by 5% fat-free milk for 2 hrs at room temperature. The primary antibody was added according to the kit instruction, shaken at room temperature for 2 hrs and then incubated at 4°C for 12 hrs. The secondary antibody (goat anti-mouse IgG, Abcam, ab205719, dilution: 1:2000; goat anti-rabbit IgG, Abcam, ab6721, dilution: 1:8000) was added to the proteins and incubated together at room temperature for 1 hr. Chemiluminescence detection was carried out using ECL reagent (Huiying, Shanghai, China). All primary antibodies (anti-SLP-2 (ab191884, 1:2000), anti-CD14 (ab183322, 1:800), anti-Cdc42 (ab64533, 1:800), anti-vascular endothelial growth factor A (VEGFA, ab1316, 1:1000), anti-Bax (ab32503, 1:1000), anti-B-cell lymphoma 2 (Bcl-2, ab59348, 1:1000), and anti-GAPDH (ab8245, 1:1000)) used in this study were purchased from Abcam.

The frozen tissues (−15°C) were sectioned (10-µm thick) using a freezing microtome. The frozen sections were washed with PBS and the membranes were permeated with 0.3% Triton X-100 (50 µl) by gavage at room temperature for 15 min. The antigen was extracted using sodium citrate solution and the sections were incubated with normal goat serum (Sangon Biotech Co., Ltd.). The sections were cultured with the antibodies against TLR4 (cat. no. ab13556, 1:500, Abcam) and CD14 (cat. no. ab183322, 1:100, Abcam) at 4°C overnight. After 1 h of incubation at room temperature, the sections were cultured with goat anti-rabbit antibody immunoglobulin G (IgG) (cat. no. ab205718, 1:2,000, Abcam) containing FITC for 1 h at room temperature. The nuclei were stained with DAPI at room temperature for 5 min and the cells were observed under a Biorevo BZ9000 fluorescence microscope (Keyence Corporation; magnification, ×200) in five randomly selected fields of view.