IHC Staining Protocol for HIPPI Protein

IHC staining for HIPPI was performed as described previously.15 (link) Formaldehyde-fixed, paraffin-embedded tissue sections were used for the IHC experiment. Tissue sections were deparaffinized, and antigen retrieval was performed in 5 mM Tris-HCl for 10 min by microwave pretreatment. Endogenous peroxidase activity was quenched with 3% H₂O₂, and serum was used to block non-specific binding sites with the Endogenous Biotin-Blocking Kit (Thermo Fisher Scientific, USA). Then, the slides were incubated with an anti-HIPPI monoclonal primary antibody (1:50, Mouse, Thermo Fisher Scientific, USA) at 4°C for 22 h, washed and then incubated with a biotinylated anti-rabbit lgG secondary antibody for 30 min at room temperature. Slides were visualized with diaminobenzidine and followed by counterstaining with hematoxylin. The representative photographs were taken using an Olympus BX50 microscope (Japan). Slides were evaluated using light microscopy and a standard semi-quantitative immunoreactivity score as described previously. By recording the percentage of positive staining (1<25%, 2=25%–50%, 3=50%–75%, 4≥75%) and staining intensity (0=negative, 1=weak, 2=moderate, 3=strong) for each sample, immunoreactivity score (IRS) (0–12) was calculated by multiplying positive staining percentage with staining intensity. Low and high expressions were defined according to the median IRS.