Peroxidase conjugated avidin biotin complex

Immunohistochemistry was performed as previously described 20 (link). 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.

Immunohistochemical staining was performed by investigators blinded to sample identity as previously described.⁵ The MEST primary antibody was purchased from Novus Biologicals (Littleton, CO, USA, AB_11006156), and antibodies against SRCIN1(AB_2881315) and RASAL1 (AB_2807382) were obtained from Proteintech (Chicago, IL, USA) and Invitrogen, respectively. Briefly, the tissue microarrays were deparaffinized and rehydrated and then heated for 15 min in 10 mM citrate (pH 6). After incubation with primary antibody at 4 °C overnight followed by corresponding biotinylated secondary antibody, the immunostaining was visualized using peroxidase-conjugated avidin-biotin complex and 3,3′-diaminobenzidine (Dako, Mississauga, ON, Canada) as the chromogen. The sections were counterstained with hematoxylin. The IHC and ISH staining intensity in the TMA was categorized: no staining as 0, weak as 1, moderate as 2, and strong as 3. Spots with 0 or 1 scoring were classified as low expression, while 2 or 3 were high expression.