Determination of protein expression in tissue

Immunohistochemistry (IHC) staining was employed to detect protein expression in tissue [13]. In short, paraffin-embedded tissue specimens were cut into 4 μm-thick sections. Tissue sections were dewaxed, rehydrated with gradient ethanol, incubated in 10 mmol/l citrate buffer (pH 6.0) and heated for 90 s. Endogenous peroxidase was blocked with 3% hydrogen peroxide (10 min). Tissue collagen was spoilt with 10% normal goat serum (10 min) for reducing non-specific binding. Rabbit polyclonal antibody for target protein (Abcam, UK) was used as primary antibody to incubate the samples for 1 h at room temperature. After washing with PBS, the samples were incubated with biotin-labeled secondary antibody (Fuzhou Maixin Biotech) and followed by streptavidin–horseradish peroxidase (HRP), both for 10 min at room temperature. Then the samples were stained with DAB (DAB-0031, Fuzhou Maixin Biotech), dehydrated and fixed with resin. Finally, the stained tissue sections were observed by experienced pathologists under inverted microscope. IHC staining was scored for each tissue section with positive staining based on the area (25%, 50%, 75%, 100%) and intensity (+ , +  + , +  + +). The final score was set to range from 1 to 4 after conversion.