Histopathological analysis
For each patient, slides stained with hematoxylin-eosin were reviewed by the same pathologist, tumor tissues were selected, and ALDH1 and CD44 immunohistochemistry was performed on these tissues. For immunohistochemical staining, 4-micron thick sections were obtained from paraffin-embedded blocks in positively charged glasses and then deparaffinized. Rehydration, blockade with hydrogen peroxide, and 20 minutes of antigen retrieval was performed with sodium citrate buffer in a microwave. Samples were incubated overnight at 2–8°C with anti-ALDH1 rabbit monoclonal antibody (ab9883, Abcam) (1:500) (5 µg/ml) and CD 44 antibody (1:50) (Santa Cruz). After incubation with antibody, samples were stained with DAB chromogen. Finally, all samples were stained with Mayer hematoxylin and washed with distilled water and PBS.
For immunohistochemical analysis, a semiquantitative evaluation was performed. The intensity and extent of staining were scored for ALDH1. Density was scored as 0: no staining; 1: weak staining; 2: moderate staining; 3: strong staining. Extent of staining was evaluated based on the percentage of positive cells. A total score was obtained by adding the density and extent scores. The evaluation of CD44 staining was based on membranous staining and was performed using the same scoring method.