Ab50276

Total protein extraction and bicinchoninic acid protein assay kits were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Polyvinylidene difluoride membranes were purchased from Merck KGaA. Antibodies against PRL-3 (ab50276; 1:400), AKT (ab8805; 1:500), p-AKT S473 (ab81283; 1:1,000), MMP-2 (ab97779; 1:1,000) and MMP-9 (ab137867; 1:1,000) were purchased from Abcam (Cambridge, UK). The antibody for β-actin (60008-1-Ig; 1:5,000) was purchased from Wuhan Sanying Biotechnology (Wuhan, China). These antibodies were used in the present study was described previously (23 (link)–26 (link)). Membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab6721; 1:3,000) or goat anti-mouse secondary antibody (Wuhan Sanying Biotechnology; SA00012-6; 1:2,000) and the western blotting protocol were performed as previously described (21 (link)). Reactive protein was detected by an enhanced chemiluminescence kit (EMD Millipore, Billerica, MA, USA). Band intensity was quantified by using the Quantity One image analysis software version 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

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.