Immunohistochemistry was performed as previously described 20. The tissue microarrays containing 202 cases of CRC tissues and 158 matched adjacent normal tissues (Shanghai Outdo Biotech, Shanghai, China) were used to analyze ARF1 expression and its correlation with clinicopathological parameters. In brief, the slides were blocked with normal serum and then incubated with anti-Ki-67 (Dako, Mississauga, ON, Canada), anti-p-ERK, or anti-ARF1 antibodies overnight at 4°C, followed by matching biotinylated secondary antibodies and peroxidase-conjugated avidin-biotin complex (Dako). 3,3′-diaminobenzidine (Dako) was used as a chromogen to visualize the immunostaining, and the sections were counterstained with hematoxylin. A scale of 1 (negative) to 2 (weak) representing low expression and a scale of 3 (moderate) to 4 (strong) representing high expression was used to grade the intensity of ARF1 and p-ERK staining.

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