Ab1823

Immunohistology was performed on formalin‐fixed and paraffin‐embedded mouse liver sections for αSMA (NBP1‐97722; Novus Biologicals; ab32575; Abcam), Col1a1 (ab34710; Abcam), Pdgfrβ (ab32570; Abcam), Sdc4 (ab24511; Abcam), Thbs1 (ab1823; Abcam), F4/80 (70076s; CST), and CD68 (ab125212; Abcam). Triple and double immunofluorescence staining using a panovue multicolor fluorescent staining kit according to the manufacturer's protocols (10079100020; Panovue). Images were acquired with a High‐content screening system (YOKOGAWA, CQ1) and a fluorescence imaging system (Nikon; Eclipse Ts2).

Cells were lysed on ice with lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP‐40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 7.5) in the presence of a protease inhibitor mixture and phosphatase inhibitors (11697498001; Roche). Protein concentrations were measured using the BCA protein assay kit (23227; Thermo Fisher Scientific). The lysates were separated by SDS‐PAGE and transferred to a PVDF membrane, followed by immunoblotting with Pdgfrβ (ab32570; Abcam), Akt (9272S; CST), p‐Akt (4060S; CST), mTOR (2972S; CST), PI3K‐p110α (4249T; CST), PI3K‐p85 (4257T; CST), p‐PI3K‐p85 (4228T; CST), Thbs1 (ab1823; Abcam) antibodies, and secondary antibodies.

For histological analysis, the following primary antibodies (Abs) were employed: anti-vitronectin Abs (ab45139, Abcam, Cambridge, MA, and MAB38751, R&D Systems, Inc., Minneapolis, MN), anti-thrombospondin 1 Abs (Ab-11, Thermo Fisher Bio-scientific, Hudson, NH, and ab1823, Abcam), anti-CD45 Abs (ab10558, Abcam, and M0701, DAKO, Carpinteria, CA, USA). Additionally, for B220⁺CD11c⁺NK1.1⁺ NK cell sorting, the following antibodies were used; PE/Cy7 anti-mouse B220 (103222, BioLegends), PE anti-mouse CD11c (117308, BioLegends), APC anti-mouse NK1.1 (108710, BioLegends), and those isotype control antibodies, PE/Cy7 Rat IgG2a, κ (400521, BioLegends), PE American Hamster IgG (12-4888-81, Thermo Fisher), APC Mouse IgG2a κ (550882, BD Biosciences).

Human ovarian carcinoma cell lines A2780 and SK-OV-3 were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively. A2780 and SK-OV-3 cells were respectively maintained in RPMI 1640 medium containing 2 mM L-Alanyl-Glutamine (GlutaMAX^TM-I, Gibco, Life Technologies, Saint-Aubin, France) and McCoy’s 5a medium modified (ATCC) supplemented with 10% heat-inactivated fetal bovine serum (FBS). ID8 mouse OC cell line was obtained from Dr. Katherine Roby (University of Kansas Medical Center) [41 (link)]. ID8 cells were maintained in high-glucose Dulbecco’s Modified Eagle’s Medium (#D6429, Sigma-Aldrich, Saint-Quentin Fallavier, France) supplemented with 4% FBS, 1% penicillin/streptomycin and insulin–transferrin–sodium selenite media supplement (ITS mix, #I1884, Sigma-Aldrich). Constitutive expression of TSP-1 and PD-L1 by ID8 cell line under unstimulated conditions was ensured by Western blot, using anti-TSP-1 (#ab1823; 1:1000 dilution) and anti-PD-L1 (#ab233482; 3 µg.mL⁻¹) antibodies from Abcam. Cells were cultured in growth medium and split every 3 to 4 days by harvesting in trypsin. Authenticated cell lines by provider were stored at early passages (< 3) in liquid nitrogen, controlled to be mycoplasma-free, and were used in the experiments for no longer than 6 months.

Cells were lysed using the RIPA buffer. The BCA method was used to detect the thrombospondin-1 (THBS1) protein concentration. Next, 25 μg of protein was loaded onto 4–20% Express PLUSTMPAGE gels (GenScript, USA) for separation, followed by transfer onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with a primary antibody overnight. Subsequently, the membranes were incubated with an HRP-conjugated secondary antibody (1:5000). The proteins were detected using the EZ-ECL kit (Biological Industries, Israel). The following antibodies were obtained from Abcam (Cambridge, UK): anti-SDC1 (ab128936), anti-SDC4 (ab74139), anti-vWF (ab134193), anti-THBS1 (ab1823), and anti-PKC-A (ab32326), anti-uPA (ab3218106), anti-VEGFA (ab1316), anti-EGFR (ab52894), anti-G6PD (ab210702), anti-GAPDH (ab8245).

Serial freezing section of FAO and v-FAO was processed as we recently described^{44 (link),49 (link)}. The freezing section at the core of FAO/v-FAO was employed to analysis the cell composite and protein expression. The following primary antibodies were used for immunofluorescence: THBS1 (ab1823, Abcam, UK), CD31 (3528, CST, USA), Collagen III (ab184993, Abcam, UK), Pan-Keratin (4545, CST, USA), COL1A1 (72026, CST, USA), COL3A1 (66887, CST, USA).

Specimens and cells were fixed with 4% paraformaldehyde for 15 min. Slides were then immersed for 10 min in 0.1% Triton X-100 and blocked with 10% normal goat serum for 30 min. The cells were then incubated overnight at 4°C with primary antibodies against rabbit anti-CD45 (1:100, Cat. No. BA3371, Boster, China), rabbit anti-CXCR4 (1:100, Cat. No. ab2074, Abcam, USA), mouse anti- thrombospondin-1 (TSP-1) (1:100, Cat. No. ab1823, Abcam, Cambridge, MA) respectively. Secondary anti-mouse antibodies (1:500, Cat. No. 4408S, CST, USA) and anti-rabbit antibodies (1:500, Cat. No. 4413S, CST, USA) were added at room temperature, the nuclei were stained with DAPI. Images were captured by fluorescence microscopy.