Immunohistochemistry (IHC)

Specimens were deparaffinized in xylene and then rehydrated in serial diluted ethanol. For antigen retrieval, the slides were boiled in 0.01M citrate buffer (pH 6.0) for 15 min in a microwave. In order to inactivate endogenous peroxidases, the slides were treated with 0.3% hydrogen peroxide for 10 min, and then blocked with 5% BSA in TBST for 1 h. The slides were then incubated with antibody against NEDD4L (13690-1-AP, Proteintech) or c-Myc (ab32072, Abcam) overnight on a rocker at 4°C. The slides were washed in TBST, then covered by secondary antibody at room temperature for 1 h. 3,3-diaminobenzidine (DAB) solution (Vector Laboratories, Burlingame, CA, USA) was dropped onto the sections to label the positively stained cells, after which the sections were counterstained slightly with hematoxylin. The intensity of immunoreactivity was graded based on an established method from a previous report (Schmidt et al., 2009): IRS (immunoreactive score) = staining intensity (SI) × percentage of positive cells (PP). The SI was categorized as 0, negative; 1, weak; 2, moderate; 3, strong. A protein with staining index ≥3 would be defined as overexpression. IRS ≥9 will be considered as a highly positive signal. The IRS score for the staining intensity ranges from 0 up to 12. The percentage of positive cells (PP) was defined as follow: 1: 0–10, 2: 11–50, 3: 51–80, 4: 81–100%. The staining score was assessed by two independent investigators. Sections of non-neoplastic brain tissues (He et al., 2012) and colon cancer tissues (Ren et al., 2020) were taken as positive control slide for NEDD4L and c-Myc staining, respectively. Matched negative controls were stained without primary antibody.