Immunostaining of RCC Tissue Sections

Immunostaining of the paraffin-embedded RCC tissue sections was performed by a similar method to that used in our previous work [36 ]. The sections were deparaffinized and rehydrated, then boiled for the antigen retrieval. The endogenous hydrogen peroxidase was blocked. After being incubated with 10% BSA, the sections were incubated with anti-C12orf59 antibody (HPA036147, Sigma, USA) used at a 1:300 dilution at 4°C overnight. After being washed in PBS, the sections were treated with a MaxVision HRP-Polymer anti-rabbit IHC Kit (Maixin Bio, Fujian, China) and stained with a DAB kit (Maixin Bio, Fujian, China). The expression of C12orf59 was assessed blindly by two independent investigators. The staining of C12orf59 was scored as the product of the staining intensity (on a scale of 0–3: negative = 0, weak = 1, moderate = 2, strong = 3) and the percentage of cells stained (on a scale of 1–5: 1 = 0%–20%; 2 = 21–40%; 3 = 41–60%; 4 = 61–80%; 5 = 81%–100%), resulting in scores ranging from 0 to 15. We defined two subgroups as follows: the low expression group (scores: 0–5) and the high expression group (scores: 6–15) [37 (link)].