Goat anti rabbit igg hrp

One hundred thirty-one GC tissues and twenty-four matched adjacent normal tissues were embedded in paraffin according to standard procedures. All tissue blocks were cut into 4 µm sections and then deparaffinized and hydrated on the basis of standard procedures. The endogenous peroxidase activity of tissue sections was blacked by using 3% H₂O₂ for 15 min at room temperature. Sections were incubated in a wet chamber with the primary antibody TAF15 (1:100, ab134916, Abcam, USA) at 4 °C for overnight, and then incubated with the secondary antibody HRP goat anti-rabbit IgG (1:100, D110058, Sangon Biotech, Shanghai, China) for 1 h at room temperature. Finally, sections were developed using diaminobenzidine and haematoxylin. The images were reviewed by two pathologists in a blinded fashion. The staining percentage was scored as follows: 0 (< 5% positive cells); 1 (5–24% positive cells); 2 (25–49% positive cells); 3 (50–74% positive cells) and 4 (≥ 75% positive cells). The staining intensity was scored as follows: 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong). The combined staining score was equivalent to the staining percentage score × staining intensity score and a combined score ≥ 4 was defined as positive expression, while a combined score < 4 was considered negative expression.

Total proteins from transfected and untransfected HepG2 cells were extracted using RIPA buffer (Vazyme Biotech, China). BCA assay (Beyotime, China) was conducted to quantify the extracted total proteins. For isolation of the protein samples, 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out followed by transfer to polyvinylidene fluoride membranes (0.45 μm) with 300 mA current for 90 min. After transfer, the membranes were blocked in 5% BSA at ambient temperature for 1 h and then incubated overnight using rabbit antibodies against GPC3 (Abcam, USA; diluted 1 : 1000) and a rabbit antibody against GAPDH (Sangon Biotech, China; diluted 1 : 5000) as an internal standard at 4°C to normalize the protein expressions. On the next day, the membranes were further incubated using goat anti-rabbit IgG-HRP (Sangon Biotech, China; diluted 1 : 5000) for 1 h and then subjected to protein visualization using enhanced chemiluminescence (Vazyme Biotech, China). The G:BOX Chemi XX9 imaging system (Syngene, UK) was used for protein visualization.