Ab24577

The antibodies to Tet1 (ab191698), Tet2 (ab94580), P2rX7 (ab48871), CD146 (ab24577), PDGFRα (ab65258), and OCN (ab10911) were purchased from Abcam (Cambridge, MA, USA). Antibodies to ALP (sc-28904), CD9 (sc-9148), and CD81 (sc-9158) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibody to Runx2 (8486) was obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody to Tet3 (20602) was purchased from Novus Biologicals (Littleton, CO, USA). Anti-CD34-PE (551387) and SCA1-PE (553108) antibodies were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD105-PE (12-1051-82), CD45-PE (25-0454-82), CD73-PE (12-0739-42), and CD90-PE (15-0902-82) antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).

TMAs were incubated during 20 min at 60 °C with antigen retrieval solution (Sigma, St. Louis, MO, USA), fixed in formaldehyde, blocked with goat serum, and incubated at 4 °C for 8 h with different antibodies to detect each cell type: LSECs with mouse anti-CD146 antibody (ab24577, Cambridge, UK), Ito cells with mouse anti-alpha smooth muscle actin antibody (ab7817, Abcam, Cambridge, UK), and Kupffer cells with mouse anti-CD68 antibody (ab31630, Abcam, Cambridge, UK). BRCA1 was detected with rabbit anti-BRCA1 antibody (orb48292, Biorbyt, Cambridge, UK). All antibodies were diluted at 1:100. Then, the samples were washed with PBS-0.3% Tween and incubated with the secondary antibody diluted 1:1000 for one minute; goat anti-rabbit Alexa 433 (Sigma, St. Louis, MO, USA) to detect BRCA1 and goat anti-mouse Alexa 594 to detect each cell type. After washing, the samples were incubated with DAPI diluted 1:1000 for 10 min.