Immunohistochemistry for Plexin-B3, Ki67, ALDH1-A1, CD31, and CD44

We performed IHC as described previously by Awaji et al. [47 (link)]. In brief, we incubated slides with primary antibodies, such as αPlexin-B3 (Santa Cruz-sc671441; 1:100), αKi67 (1:100; Santa Cruz-sc15402), αALDH1-A1(1:100; sc374149), αCD31 (1:50, Abcam,-ab28364), and αCD44 (1:500; Abcam-ab157107), in antibody diluent overnight at 4 °C. The next morning, we washed the slides and incubated with biotinylated anti-rabbit or anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 45 min. Images were captured through the Nikon microscope using NIS Element software.
IHC scoring was performed according to the following criteria: percentage of positive cells on the slides was as follows: 0 (negative), 0.1 (1–10% of cells positive), 0.2 (11–20% of cells positive), and 0.3 (20–30% of cells positive), and so forth. Furthermore, the intensity was designated as weak (1 point), moderate (2 points), strong (3 points), or very strong (4). The IHC composite score was calculated by multiplying the extent of positive cells with intensity. Two independent observers examined each slide, and their observations were positively correlated with each other. Average scores were used for analysis, and if the two observers significantly differed in their scoring, a third observer examined the slide.