Nbp2 30332

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.

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 antibodies recognizing human CD133 (MBS462020; MyBioSource), Nanog (ab80892; Abcam), and p-Akt1/2 (#9271; Cell Signaling) in a solution of PBS with 1% BSA and 0.1% Triton X-100 at 4 °C overnight. Staining was visualized using secondary antibodies tagged with Alexa Fluor 488 (A32766, A10042) and Alexa Fluor 594 (A32744, A32754), from Thermo Fisher, with nuclear counterstaining using DAPI. Images were collected on an inverted confocal microscope (Leica Microsystems) and processed using Imaris 7.6. Five fields were examined for each sample.