CD133 (Affinity Biosciences, AF5120, Polyclonal, 1:100) protein expression in specimens obtained before NCRT in 169 LARC patients was measured using the immunohistochemical streptavidin-biotin complex method (Fig. 3e, f, g, h) [18 (link)]. Phosphate-buffered saline (PBS) was used as the negative control and the image of the positive control from GE Healthcare Life Sciences. Immunoreactivity was scored by semi-quantitative analysis, and the fields were randomly selected in five directions (up, center, down, left, and right) under high magnification (× 400). The color was determined based on the intensity score as follows: 0 (no staining), 1 (light yellow), 2 (brown), and 3 (deep brown). The percentage of positive cells was scored as 0 (< 5%), 1 (5–25%), 2 (25–50%), 3 (50–75%), and 4 (> 75%). The mean value was calculated for each case with the aforementioned scoring methods and the final score was obtained by multiplying these two scores. All analyses were performed in a double-blinded manner.
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