Bs 0649r

To verify the difference in the expression of LPAR5 between tumor tissues and normal tissues and the relationship between LPAR5 and immune cells, immunohistochemical and immunofluorescence staining were performed according to the manufacturers’ protocols. Tumor tissue slides and paraneoplastic tissue slides of osteosarcoma were obtained from the Department of Pathology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Anti-CD8 antibody (ab178089, 1:400, Abcam) was used to label CD8⁺ T cell. Anti-CD68 antibody (bs-0649R, 1:400, Bioss), anti-CD163 (ab182422, 1:400, Abcam) and anti-CD206 (60143-1-lg, 1:400, Proteintech) were used to label macrophages. Anti-LPAR5 (bs-15366R, 1:400, Bioss) was used to detect the expression of LPAR5. The immunohistochemical staining images were captured by EVOS FL Auto automatic microscopic imaging system (Life Technologies, US). The immunofluorescence staining images were captured by a high-resolution slide scanning system (Pannoramic MIDI; 3DHISTECH, Hungary). Five high magnification fields were randomly selected for each slide and subsequently averaged for quantitative analysis. We used t-test to test the significance. The study has been approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (TJ-IRB20211241).

For Western blot, the primary antibodies were as follows: rabbit anti AXL (1:500, bs-5180R, Bioss, Beijing, China), rabbit anti GAS6 (1:500, bs-7549R, Bioss), mouse anti GFAP (1:1000, MAB360, Millipore), rabbit anti CD68 (1:500, bs-0649R, Bioss), mouse anti ABCA1 (1:500, ab18180, Abcam, Cambridge, United Kingdom), rabbit anti IL-1β (1:500, ab9722, Abcam), rabbit anti TNFα (1:500, ab6671, Abcam), rabbit anti IL-6 (1:500, ab7737, Abcam), rabbit anti STAT1 (1:1000, 14994, CST), rabbit anti phospho-STAT1 (Tyr701) (1:1000, 9167, CST), and β-actin (1:1000, ab8227, Abcam). For immunofluorescence, the following antibodies were used: mouse anti GFAP (1:500, MAB360, Millipore), mouse anti NeuN (1:500, ab104224, Abcam), goat anti Iba-1 (1:500, ab5076, Abcam), rabbit anti AXL (1:500, bs-5180R, Bioss), rabbit anti GAS6 (1:500, bs-7549R, Bioss), and mouse anti ABCA1 (1:500, ab18180, Abcam). For flow cytometry, ACSA-2 antibody, anti-mouse, APC (1:50, 130-117-53, Miltenyi Biotec) and isotype control antibody, rat IgG2b, APC (1:50, 130-123-825, Miltenyi Biotec).