Mtn 12

O.C.T-embedded tumor sections (7 μm-thick) were incubated with blocking solution (PBS, 5% goat serum (G9023, Sigma-Aldrich), 5% rat serum (R9759, Sigma-Aldrich), 1% FCS) for 1 hour at room temperature before incubation with primary antibody overnight at 4°C [anti–tenascin-C from Sigma-Aldrich (MTn-12)) at 10 μg/mL ; anti–F4/80-PE (BM8) and anti–CD206-APC (C068C2) from Biolegend] both at 1/100. The slides were then incubated with secondary antibody for 1 hour at room temperature (anti-IgG1-AF488, Biolegend) at 1/200, counterstained with DAPI (Thermofisher), and embedded with Fluoromount-G™ (Invitrogen). The fluorescent signal was analyzed with a Zeiss Axio Imager microscope. Five random 20x-fields per section were analyzed by ImageJ (National Institutes of Health, USA) to assess the infiltration of macrophages, as well as their spatial distribution between the tumor nest and the stroma (1 section per tumor, 5 tumors per group). Macrophage subsets and tenascin-C localization in the tumor were also analyzed and displayed as a linescan using FiJi software.

NT193 cells were lysed using RIPA buffer with protease inhibitor cocktail I (12801640; Fisher Scientific) at 1:1000, and the protein concentration was determined using a BCA assay (Thermofisher). 5% polyacrylamide gels were prepared, and protein (20 μg/well) was loaded in Laemmli buffer diluted 1/6 (stock solution: 2.5 mL of 0.5 M Tris-HCl pH 6.8; 1.2 g SDS; 2.5 mL β-mercaptoethanol; 5 mL glycerol; 5 mg bromophenol blue). Separated proteins were transferred to a nitrocellulose membrane using a TransBlot Turbo transfer machine (Biorad). Membranes were blocked for one hour in blocking buffer (PBS, 0.05% Tween20, 5% BSA) and incubated overnight with primary antibodies recognizing tenascin-C (MTn-12; Sigma-Aldrich) at 1/4000, vimentin (V5255, Sigma-Aldrich) at 1/2000 or beta actin (937215; Enzo Life Science) at 1/1000, and then for one hour with secondary antibody (anti-rat IgG A5795; Sigma Aldrich, anti-mouse IgG P0260; DAKO) at 1/20000 in blocking buffer. Proteins were detected with ECL Amersham reagent using Amersham Hyperfilm™ ECL (VWR International) and a Curix 60 film processor (AGFA).