Immunohistochemical Analysis of FAM129A

The paraffin blocks of tumorous and paracancerous renal tissues were cut into 4-μm slices. The slices were boiled in citrate buffer (0.01 M, pH 6.0) in a microwave oven using high power for 4 min, and cooled to RT. After doing this four times, the slices were washed clean with PBS for 3 × 5 min, blocked in 3% H₂O₂ for 20 min and in 10% nonimmune goat serum for 15 min at RT, and incubated with FAM129A antibody (1:200) at 4 °C overnight. The tissue slices were then warmed at 37 °C for 30 min, treated with biotin-streptavidin HRP detection kit (ZSGB-BIO, China), and imaged with 3,3′-diamino-benzidine (DAB) development kit (ZSGB-BIO, China) under an upright light BX3-CBH microscope (Olympus, Japan).
The degree of IHC immunoreactivity was judged by multiplying the staining immunoreaction intensity (Score I) and the DAB positive staining quantity (Score II) of tumor cells as previously reported^{33 (link)–35 (link)}. Score I was classified into four grades, 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). Based on DAB positively stained cells, Score II was rated as 0 (none), 1 (1–10% cells per field), 2 (10–50%), 3 (51–75%) and 4 (>76%). Immunoreactivity degrees with scores of 0–2, 3–5, 6–8 and 9–12 were considered as negative (−), weak (+), moderate (++) and strong (+++) expression of FAM129A. IHC assays were separately scored by two experienced pathologists.