Aldh1

Four micrometer sections from FFPE tumor tissue blocks were taken using a microtome (Leica Biosystems, Germany), and the sections were mounted on 3-aminopropyl triethoxysilane (APES)-coated slides. Immunohistochemistry was performed using an auto-immunostainer instrument, Ventana Benchmark XT (Ventana Medical Biosystems, USA), and Ventana reagents according to the manufacturer's protocol. Briefly, the protocol includes de-paraffinization with the EZ prep solution and antigen retrieval using the cell conditioning solution (CC1) at 95°C, followed by incubation with Optiview peroxidase inhibitor for 4 minutes, Optiview HQ linker for 8 minutes, 100 μl of the respective primary antibodies of ALDH1 (polyclonal dilution 1:100, Invitrogen), CD133 (9G9D8, 1:200 My Biosource), and CD166 (polyclonal, 1:1000, Invitrogen) each for 32 minutes and incubation with an Opti view Horseradish peroxidase (HRP) multimer for 8 minutes. The antigenantibody complex was detected by 3,3'-diaminobenzidine (DAB), counter-staining with hematoxylin for 8 minutes and post-counter-staining with a bluing reagent for 4 minutes. The slides were later mounted using DPX and xylene. Slide images were captured using an inverted trinocular microscope (Carl Zeiss, Axio vert ALFL).