4 6 diamidino 2 phenylindole dapi 28718 90 3

Commercially available paraffin-embedded tissue array slides containing 79 human sarcomas and 4 human normal tissues (NBP2-30332, Novus Biologicals, and T242, US Biomax, Inc., USA) were purchased. Sections were deparaffinized, then incubated with anti-human CD133, PIK3R3 (SR44-06; Novus Biologicals), and Nanog (ab80892; Abcam) in a solution of PBS with 1% BSA and 0.1% Triton X-100 at 4 °C overnight. Staining was visualized using anti-mouse Alexa Fluor 488 (A-11029; Thermo Fisher), and anti-rabbit Alexa Fluor 594 (A-11037; Thermo Fisher), with nuclear counterstaining using 4′,6-diamidino-2-phenylindole (DAPI, 28718-90-3; Sigma-Aldrich) and imaging on an inverted confocal microscope. Images were processed using Imaris 7.6.

Spheroid cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Following cell fixation, cells were incubated with antibodies recognizing CD133 (MBS462020; MyBioSource), Nanog (#4893; Cell Signaling), and γ-H2AX (05-636; Millipore) in a solution of PBS with 1% FBS and 0.1% Triton X-100 at 4 °C overnight. Cells were then stained with secondary antibodies tagged with Alexa Fluor 488 (A-11029) or Alexa Fluor 568 (A-11061), both from Thermo Fisher. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI, 28718-90-3; Sigma-Aldrich). Stained cells were visualized on an inverted confocal microscope (Leica Microsystems) and images were processed using Imaris version 7.6 (Bitplane). Three samples were examined for each group, and each sample was visualized for five fields.