Af5120

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.

The paraffin-embedded sections were dewaxed and rehydrated. Following incubation in H₂O₂, the sections were blocked with 5% normal goat serum and hybridized with primary antibodies against HOXA5 (Bs-5713R, Bioss, China), Ki-67 (AF0198, Affinity, China), and CD133 (AF5120, Affinity, China) at 4 °C overnight. The sections were then co-incubated with horseradish peroxidase-conjugated secondary antibodies (IgG, #31460, Thermo Fisher Scientific, China) for 1 h at 37 °C. Specimens were detected using a DAB solution in the dark, counterstained with hematoxylin, and mounted. Images were obtained using an optical microscope (Olympus, Tokyo, Japan).

Sections were incubated with anti-CD34 (AF5149, Affinity Biosciences, USA), anti-CD133 (AF5120, Affinity Biosciences, USA), and anti-CD31 (GB13428, ServiceBio, China) at 4°C overnight. Then, sections were immunoblotted with fluorescence-conjugated secondary goat anti-rabbit antibodies. The nucleus was stained with 4’6-diamidino-2-phenylindole (DAPI). Finally, images were captured under a fluorescence microscope.