Goat anti mouse igg alexa fluor 488 or 546

Cells were fixed with 4% (v/v) paraformaldehyde (PFA; Sigma) and stained according to a previously described protocol (Miranda et al., 2015 (link)). MAP2 (Sigma, 1:500), glial fibrillary acidic protein (GFAP, Abcam, 1:200), Synaptophysin (SYN; Abcam, 1:200), ZO-1 (Novex, 1:100), SOX2 (R&D, 1:200), PAX6 (Covance, 1:400), NESTIN (R&D, 1:400), Ki-67 (Abcam, 1:100), HuC/D (Thermo Fischer Scientific, 1:100), activated CASPASE3 (pCASP3, Cell Signaling, 1:400), were used as primary antibodies whereas goat anti-mouse IgG Alexa Fluor–488 or 546 (1:500, Invitrogen), goat anti-rabbit IgG Alexa Fluor–488 or 546 (1:500, Invitrogen) were used as secondary antibodies. Fluorescence images were acquired with Zeiss LSM 710 Confocal Laser Point-Scanning Microscope using 20× and 63× objectives and integrated density were calculated for each channel using ImageJ software. The ratio between integrated density for the marker of interest and nuclear counterstaining with DAPI was calculated for each image. For each staining, the same acquisition settings were applied for all images.

Primary antibodies used for immunofluorescence microscopy and flow cytometry included OCT4 (1:750) and NANOG (1:5000) (Milipore), SOX2 (1:1000) (R&D Systems), SSEA4 (1:135), SSEA4-PE (1:10), TRA-1-60 (1:135), TRA-1-60-PE (1:10) and TRA-1-81 (1:135) (StemGent). Secondary antibodies included goat anti-mouse IgG Alexa Fluor– 488 or 546 (1:500 or 1:1000), goat anti-rabbit IgG Alexa Fluor 546 (1:1000)–Invitrogen; and isotypes used for control in flow cytometry tests included anti-mouse IgM-PE (Miltenyi Biotec) and anti-mouse IgG-PE (1:10) (StemGent). Immuncytochemistry against markers from the three germ layers was performed using antibodies against alpha smooth muscle actin (α-SMA; mouse: 1:1000; Dako), neuron-specific class III β-Tubulin (TUJ1; mouse: 1:20 000; Covance) and SOX17 (mouse: 1:1000; R&D Systems), for the mesoderm, ectoderm and endoderm, respectively. Cardiomyocyte marker was Troponin T cardiac isoform antibody (13–11) (cTNT; mouse: 1:500; Thermo Scientific). Neural progenitor cell markers were NESTIN (mouse: 1:1000; R&D Systems) and paired box gene 6 (PAX6; rabbit: 1:1000; Covance).