Immunohistochemical staining was performed on 4 μm tissue sections using an EnVision™ Kit (DAKO, Denmark). After deparaffinized, rehydrated in graded ethanol, antigen retrieval and blocking, the slides were incubated overnight at 4 °C in a humidified chamber with anti-ALKAL1 antibodies (Thermo Fisher, PA5-55591), Vimentin (sc-373717), Ki67 (27309-1-AP, Proteintech) and caspase 3 cleavage (Asp175, 9661, Cell Signaling Technology) diluted 1:50 in PBS. Scores given by two independent Pathologists were averaged for further comparative evaluation of ALKAL1 expression. The details of scoring criteria were described in our previous study 26 (link), 27 . According to this method, ALKAL1 expression in colorectal cancer samples was evaluated by the staining index, with scores of 0, 1, 2, 3, 4, 6, 8, 9 or 12. High or low expression of ALKAL1was stratified according to the following criteria: SI ≤ 4 was defined as tumors with low expression of ALKAL1, and SI ≥ 6 was defined as tumors with high expression of ALKAL1.
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