Af1738

The lung and breast tissues were lysed in RIPA buffer (Applygen, Beijing, China) containing a phosphatase inhibitor. Human FSTL1 antibody (AF1694; R&D Systems, Minneapolis, USA), Mouse FSTL1 antibody (AF1738; R&D Systems), and antibodies recognizing the following proteins were used: Phospho-p44/42 mitogen-activated protein kinase (MAPK) (#4370; Cell Signaling Technology, Boston, USA), β-actin (#4970; Cell Signaling Technology). Western blot analyses were performed following standard protocols as described previously [20 (link)].

Human breast cancer samples were obtained from the Cancer Hospital of Huanxing Chaoyang District Beijing and were fixed in 4% paraformaldehyde (PFA) for 24 to 48 hours. Mouse metastatic lung tissue was dissected and fixed in PFA for 48 hours, and then stored in 70% alcohol. All samples were embedded in paraffin, and 5 µm sections were obtained using a Paraffin slicer (RM2235; Leica, Heidelberg, Germany). For hematoxylin and eosin (H&E) staining, lung tissue was stained with H&E. For immunohistochemistry, sections were deparaffinized and rehydrated, then incubated with primary antibodies (FSTL1, AF1694 and AF1738; R&D Systems) at 4℃ overnight, followed by a horseradish peroxidase secondary antibody (donkey anti-goat IgG H&L, ab97110; Abcam, Cambridge, UK) at 37℃ for 1 hour. Samples were developed using a diaminobenzidine solution (ZsBio, Beijing, China) for 2 minutes at room temperature. Sections were then dehydrated and affixed to coverslips.