Anti sox2

Immunostaining was performed using anti-OCT4, anti-SEEA4, anti-TRA-1-60, anti-SOX2 (Thermo Fisher Scientific, Waltham, MA, USA), anti-NKX2.5, anti-TNNT2/cTNT, anti-alpha-actinin (Sigma-Aldrich, St. Louis, MO, USA, A7811), and MYL2 (ProteinTech, Rosemont, IL, USA, 10906-1-AP) as primary antibodies. The procedure was performed on a 24-well culture dish with 60–70% cell confluence. The cells were washed with PBS twice, followed by fixing with 4% formaldehyde in Dulbecco’s PBS (DPBS) for 15 min. After additional washing with PBS, the cells were permeabilized by 1% saponin in DPBS for 5 min and blocked with 3% BSA in DPBS for 30 min at room temperature. The cells were incubated with the designed primary antibody overnight at 4 °C. Then, the cells were washed thrice with PBS and incubated with the designed secondary antibodies for 1 h. The chamber was washed thrice with PBS, and nuclear counterstaining was carried out with NucBlue™ Fixed Cell ReadyProbes™ Reagent (4′,6-diamidino-2-phenylindole (DAPI), Thermo Fisher Scientific, Waltham, MA, USA) for 1 min. After washing with PBS twice, images were captured with a Zeiss Axioplan 2 Imaging microscope with Plan-NEOPLUAR 10×/0.75 objective lens.

Immunofluorescent detection of SOX2 in mammospheres was performed as described in Leis et al.^{18 (link)}: slides were fixed in methanol and then washed 3 times in washed solution (PBS-BSA 0.1) for 5 min and permeabilized with PBS-BSA 0.1 + 0.3 triton X100 for 30 min at RT. Following permeabilization, the slides were incubated in blocking solution (PBS-BSA 0.1 + Normal Goat Serum) and the corresponding primary antibody (anti-SOX2, cat # PA1-16968, Thermo) ON at 4C. Next, the slides were washed 3 times in washing solution for 5 minutes and incubated with the corresponding secondary antibody (anti-rabbit Alexa Fluor (TM) 555 cat # A31572, Life Technologies).

Primary antibodies and dyes employed in this study were anti-nestin (Abcam, cat. no. ab22035, 1:1000), anti-Sox2 (ThermoFisher Scientific, cat. no. 149811-82, 1:200), anti-Pax6 (Biolegend, cat. no. PRB-278P, 1:100) and anti-TOMM20 (ThermoFisher Scientific, cat. no. PA5-52843, 1:100). Secondary antibodies were goat anti-mouse IgG H&L (Alexa Fluor 555) (Abcam, cat. no. ab150118, 1:500) and goat anti-rabbit IgY H&L (Alexa Fluor 488) (Abcam, cat. no. ab 150077, 1:500). Nuclei were visualised using Hoechst 33342 stain (ThermoFisher Scientific, cat. no. 62249, 1:10,000).