Immunohistochemistry (IHC) was used to assess the protein expression of six CSC markers (CD133, CD44, ALDH1, SOX2, OCT4, and Nanog) and two EMT markers (E-cadherin and Snail-1). An automated immunostainer (Benchmark Ventana, Tucson, AZ) was used to stain tissue sections following the manufacturer's recommended procedure. The primary antibodies used against the follows: CD133 (1:200; Spring Bioscience, Pleasanton, CA), CD44 (1:200; Thermo Scientific, Fremont, CA), ALDH1 (1:100; BD Biosciences, San Diego, CA), SOX2 (1:100; Cell Signalling, Beverly, MA), OCT4 (1:100; Cell Marque, Rocklin, CA), Nanog (1:100; Epitomics, CA), E-cadherin (1:100; BD Biosciences, San Jose, CA) and Snail-1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA). IHC results were graded semiquantitatively based on the percentage of cells stained and the intensity of staining [34 (link)]. Briefly, the staining intensity was graded as weak (1+), moderate (2+), or strong (3+) and was multiplied by the percentage of positive cells. The total score was then classified as follows: 0-100 = grade 1, 101-200 = grade 2, and 201-300 = grade 3. Grade 2 or 3 tumors were considered to be positive for CSC markers and E-cadherin [35 (link)]. For Snail-1, tumors were considered positive when at least 10% of all tumor cells were immunoreactive [36 (link)].
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