For IHC, the paraffin sections were deparaffinized and dehydrated by xylene and a series of graded ethanol according to the procedure described in our previous study [9 (link)]. Paraffin-embedded ccRCC tissues, NATs, orthotopic tumor tissues, and mouse metastasis lungs were subject to IHC analysis to determine the protein expression using antibodies against SOX9 (ab185966, 1:1000, Abcam, Burlingame, CA, USA), FUS (ab243880, 1:300, Abcam), Ki67 (ab92742, 1:500, Abcam), and α-SMA (ab32575, 1:100, Abcam) at 4 °C overnight. Subsequently, the tissues were incubated with secondary antibody (GB23303, 1:400, Servicebio, Wuhan, China) for 1 h at room temperature. Finally, the sections were fixed with neutral balata and photographed by a fluorescence microscope (Olympus, Tokyo, Japan). The staining area score was measured as 0, < 5%; 1, 5–25%; 2, 25–50%; 3, 50–75%; and 4, > 75%. The staining intensity score was measured as 0, no staining; 1, weak staining; 2, moderate staining; and 3, intense staining. The total staining score was determined by two independent pathologists, and the total score was calculated by combination of staining intensity and area, where samples with a score ≥ 6 were considered to have high expression, while those with a score < 6 were considered to have a low expression [25 (link)].
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