Immunohistochemical Scoring of Protein Expression

Immunohistochemistry was routinely conducted as previously described [27 (link), 28 (link)]. After antigen retrieval, the slides were incubated with the following primary antibodies: NSE (1:100; Abcam), rabbit anti-ALDH1A1 polyclonal antibody (1:200; Abcam), and NBL1 (1:200; Affinity) overnight at 4 °C. Immunohistochemical staining was performed using diaminobenzidine (DAB). After counterstaining with hematoxylin, the sections were dehydrated and sealed. Staining scores were assessed by two different individuals (ZQZ and PLZ). The percentage and intensity of staining were evaluated and multiplied to obtain the final scores. The principle of immunohistochemical scoring is mainly based on staining intensity (0–3 points) and staining area (0–3 points). Finally, the product is the total score. Dyeing intensity was as follows: 0 points for no dyeing, 1 point for light yellow, 2 points for yellow or brown, 3 points for brown or tan; the stained area was as follows: 0 points for ≤5%, 1 point for 5–25%, 2 points for 25–50%, and 3 points for ≥50%. An immunohistochemical score of ≤3 indicated a low expression group, and a score >3 indicated a high expression group [29 (link)].