TMA construction and immunohistochemistry were conducted following a two-step method as detailed previously (21 (link)). Briefly, the primary antibody (polyclonal rabbit anti-human PRL-3, ab50276, ABCAM, Cambridge, UK) was diluted 1:400 in phosphate-buffered saline (Hyclone, Logan, Utah, USA) containing 1% bovine serum albumin (Gibco, USA).
After incubating with the appropriate biotin-conjugated secondary antibody, the sections were stained with DAB Horseradish Peroxidase Color Development Kit (Beyotime, China). The intensity of the cytoplasmic and nuclear membrane immunostaining for PRL-3 was graded as follows: 0, no immunostaining; 1, immunostaining detected in 25% of tumor cells; 2, moderate immunostaining marked in 25–50% of tumor cells; and 3, strong and diffuse immunostaining observed in over 50% of tumor cells. Subsequently, a score of 0 to 3 was considered negative, weakly positive, moderately positive, and strongly positive. Two independent pathologists evaluated the immunohistochemical staining under a light microscope.
After incubating with the appropriate biotin-conjugated secondary antibody, the sections were stained with DAB Horseradish Peroxidase Color Development Kit (Beyotime, China). The intensity of the cytoplasmic and nuclear membrane immunostaining for PRL-3 was graded as follows: 0, no immunostaining; 1, immunostaining detected in 25% of tumor cells; 2, moderate immunostaining marked in 25–50% of tumor cells; and 3, strong and diffuse immunostaining observed in over 50% of tumor cells. Subsequently, a score of 0 to 3 was considered negative, weakly positive, moderately positive, and strongly positive. Two independent pathologists evaluated the immunohistochemical staining under a light microscope.
