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.