Ab64693

Opal 7-colour kit (PerkinElmer, NEL811001KT) was used for multiplex IHC. Four micrometers of FFPE sections were dewaxed and rehydrated. In the first round antigen was retrieved with a pressure cooker (EDTA pH 8.0) at 125 °C for 3 min. Slides were cooled to room temperature (RT), washed with TBST/0.5% Tween (3 times, 5 min) and incubated with H₂O₂ (3%) for 10 min. Slides were washed and blocked with blocking buffer (Dako, Glostrup, Denmark) for 10 min. Primary antibody, CD163 (Cell Marque, MRQ-26, 1:500, dye540), was incubated at RT for 30 min. Slides were washed and an HRP-conjugated secondary antibody was incubated at RT for 10 min. TSA dye (1:50) was applied for 10 min after washes. This was repeated five more times using the following antibodies, CD68 (Leica Biosystems, 514H12, 1:100, dye570), CD206 (Abcam, Ab64693, 1:6000, dye620), IRF8 (Santa Cruz, E-9, 1:3000, dye650), PDL1 (Spring Bioscience, SP142, 1:2000, dye520), and multi-cytokeratin (Leica Biosystems, NCL-L-AE1/AE3, 1:200, dye690). For second and subsequent rounds antigen retrieval was performed in EDTA (pH 8.0) buffer using a microwave (100–150 mW, 15 min). Nuclei were stained with DAPI (PerkinElmer) and mounted with medium (HardSet, Vectashield). Secondary antibodies anti-rabbit (PerkinElmer, NEF812001EA) or anti-mouse (PerkinElmer, NEF822001EA) were used at a 1:1000 dilution.

Total protein was extracted from RAW 264.7 cells using the same procedures as described in detail elsewhere (Santa et al., 2016 (link)). Briefly, raw cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo, 89901) containing proteinase inhibitors and phosphatase inhibitors (KeyGEN BioTECH, KGP250). The protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, 23,225). Subsequently, the phenol–ethanol supernatant was mixed with isopropanol to isolate proteins. Equal amounts of protein and supernatants were separated by 12.5 or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.45 or 0.20 μm-pore polyvinylidene fluoride membranes (Merck Millipore, IPVH00010 or ISEQ00005). Membranes were incubated overnight with antibodies against CD163 (Abcam, ab182422), CD206 (Abcam, ab64693), PDGF-BB (Santa Cruz, sc365805), MMP9 (Abcam, ab38898), and β-actin (Cell Signaling echnology, 2118). The membranes were then incubated with peroxidase-conjugated goat antirabbit immunoglobulin G (IgG) (h + l) secondary antibody (1:5,000) for 1 h. Protein signals were detected using an enhanced chemiluminescence kit (Cat. WBKLS0500, Millipore), and Western blot bands were examined and analyzed with a chemiluminescence instrument (Guangzhou Ewell Bio Technology Co. Ltd., China).