Tissue microarray (TMA) and immunohistochemistry (IHC)

TMAs were constructed by Shanghai Biochip Co., Ltd. (Shanghai, China). Pathological types of all samples were reviewed by hematoxylin and eosin staining, in accordance with protocols from a previous study (25). IHC of paraffin sections was performed according to a protocol from a previous study (27). In brief, paraffin sections were deparaffinized by heating at 65°C for 2 h, subsequently washed with xylene and rehydrated in ethanol. After antigen retrieval was performed by incubating in 10 mmol/l Citrate Sodium Buffer (pH 6.0; Yesen) and the slides boiled in a microwave, slides were incubated in 0.3% H₂O₂ for 15 min to block endogenous peroxidase activity at room temperature. Slides were rinsed in PBS and immersed in 5% BSA for 1 h at room temperature to block nonspecific binding sites. Subsequently, the sections were incubated with primary antibody, rabbit anti-human FUS/TLS (dilution, 1:5,000; catalog no. 11570-1-ap; ProteinTech Group, Inc.; Chicago, IL, USA), overnight at 4°C. The following day, samples were washed three times with PBS. After 30 min of incubating slides in horseradish peroxidase labeled secondary antibody (GTVision III immunohistochemical kit; cat. no. GK500705; Gene Tech, Shanghai, China) at room temperature and washing in PBS buffer, the slides were stained with 3,3′-diaminobenzidine (DAB)-H₂O₂ under a microscope for 2 min at room temperature, and hematoxylin was used to counterstain the nuclei at room temperature for 40 sec. Finally, the sections were dehydrated and covered with glass microscope glass using neutral resins.