Immunohistochemical Evaluation of EDG2 Expression

Immunohistochemistry (IHC) staining was carried out as described previously [19 (link)]. Briefly speaking, 4 μm-thick specimen slides for IHC staining were deparaffinized, and peroxidase was quenched with methanol and 3% H2O2 for 15 min. All sections were boiled with pressure in citrate buffer for 3 min and blocked overnight at 4°C. After incubated for 4 h with primary rabbit monoclonal anti-human EDG2 antibody (ab166903, Abcam, UK) at 1:100 dilution, the slides were washed with phosphate buffered saline (PBS), incubated with HRP-conjugated goat anti-rabbit antibody (ab6721; Abcam, UK) for 15 min at 1:100 dilution, and washed again with PBS. The staining of the slides was performed with the avidin-biotin-peroxidase complex. Finally, the sections were visualized with diaminobenzidine and counterstained with hematoxylin.
The scoring system for IHC staining was presented previously [16 (link)]. Staining intensity was divided into four grades: 0, none; 1, weak; 2, moderate; 3, strong. The percentage of specifically positive staining tumor cells was classified with the following grades: 0 (<5%), 1 (6%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (>75%). The final score was expressed by multiplying the staining intensity and the percentage of specifically positive staining tumor cells.