A6276

ccRCC tissues and adjacent normal tissues were sequentially fixed in formalin, dehydrated and embedded in paraffin. Subsequently, IHC was conducted by incubating tissue sections (4 µm) with a primary rabbit PLIN2 polyclonal antibody (1:100; A6276; ABclonal Biotech Co., Ltd., Wuhan, China) overnight at 4°C. Subsequently, after washing three times with PBS, the sections were incubated with goat anti-rabbit secondary antibody (1:200; GB23303; Servicebio, Inc., Woburn, MA, USA) at room temperature for 2 h.

Tissues and cells were lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing a protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN, USA) and 1 mM phenylmethylsulfonyl fluoride. Subsequently, the protein concentrations were measured using a bicinchoninic acid kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Total proteins (30 µg) were separated by 10% SDS-PAGE and were then transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Bedford, MA, USA) at 90 V for 90 min. The PVDF membranes were blocked in PBS containing 5% nonfat milk for 1 h at room temperature, and were then incubated with primary antibodies against PLIN2 (1:1,000; A6276; ABclonal Biotech Co., Ltd.) and GAPDH (1:3,000; BM3876; Wuhan Boster Biological Technology, Ltd., Wuhan, China) overnight at 4°C. Subsequently, the membranes were incubated with secondary antibodies (1:3,000; GB23303; Servicebio, Inc.) for 2 h at room temperature. Finally, the proteins were visualized using ChemiDoc-XRS⁺ (Bio-Rad Laboratories, Inc., Hercules, CA, USA).