Mouse anti cpm

Undifferentiated hiPSCs and AECs were rinsed with PBS and fixed with 4% paraformaldehyde (Alfa Aesar) for 20 min at room temperature. Cells were permeabilized with 0.5% saponin (Sigma) and blocked with 10% normal donkey serum (Jackson Immuno., USA) diluted with 1% BSA in PBS. The following primary and secondary antibodies were used: rabbit anti-OCT4 (1:200, BD Pharmingen), rabbit anti-NKX2.1 (1:250, Abcam), mouse anti-CPM (1:500, Abcam), mouse anti-EPCAM (1:400, Santa Cruz), Alexa 594 (Invitrogen) donkey anti-mouse IgG(H + L), Alexa 488 (Invitrogen) donkey anti-rabbit IgG(H + L) and Alexa 594 (Invitrogen) donkey anti-rabbit IgG(H + L). Nuclei were counterstained with Fluoroshield with DAPI (Sigma) for 5 min, and fluorescent images were captured with a fluorescence microscope (IX-51, Olympus, JAPAN).

Undifferentiated PSCs and AECs were rinsed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma) for 20 min at room temperature. After washing with PBS, cells were permeabilized with 0.5% saponin (Sigma) in PBS containing 1% bovine serum albumin for 10 min and then blocked with 10% normal rabbit or donkey serum for 30 min. The following primary and secondary antibodies were used: goat anti-OCT3/4 (Santa Cruz), mouse anti-CPM (Abcam), rabbit anti-NKX2.1 (Abcam), mouse anti-EPCAM (Santa Cruz), rabbit anti-goat Alexa Flour 594 (Invitrogen), goat anti-mouse Alexa Fluor 594 (Invitrogen), donkey anti-mouse Alexa Fluor 594 (Invitrogen), and donkey anti-rabbit Alexa Fluor 488 (Invitrogen). Nuclei were stained with prolong gold DAPI with antifade (Invitrogen). Immunofluorescence signals were observed and photographed with an Olympus IX51 microscope equipped with a photometrix Cool Snap HQ2 camera using Image-Pro 3DA vs. 6.0. software.