Mouse monoclonal anti igfbp5

The parental and transfected cells were washed with prechilled PBS and lysed in 1 × RIPA buffer (Sigma, St. Louis). The protein concentration was measured by the Bradford method with BSA (Sigma) as the standard. Equal amounts of protein extract (50 μg) were denatured with 8–10% SDS-PAGE, transferred to PVDF membranes (Millipore), and blocked with 5% non-fat milk in TBST (0.1 M, pH 7.4). Protein abundance of GAPDH (1:500; Abgent) served as a control for protein loading. Each sample was treated with rabbit polyclonal anti-ADAMTS5 (1:250; Abcam) or mouse monoclonal anti-IGFBP5 (1:250; Abcam) primary antibodies at 4 °C overnight. Membranes were incubated with secondary antibodies, HRP-conjugated rabbit/mouse anti-IgG (LI-COR Biosciences), diluted at 1:16,000 in TBST, for 2 h at room temperature. Protein bands were detected by the Odyssey infrared imaging system (LI-COR Biosciences).

CRC and normal colorectal tissues were sectioned into 4-μm thickness slides and were incubated with rabbit polyclonal anti-ADAMTS5 (1:50; Abcam) or mouse monoclonal anti-IGFBP5 (1:100; Abcam) overnight at 4 °C in a humidified container. Detection was determined by a Non-biotin Horseradish Peroxidase Detection System and DAB substrate (Dako).
All samples were observed by two pathologists. If there was a disagreement, the observers would re-examine and reach a consensus. For scoring expression of antibodies, both the intensity and extent of immunopositivity were considered. The dominant staining intensity in tumors was scored as follows: 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. The extent of positive staining tumor cells was scored as follows: <25% = 1, 25–50% = 2, 51–75% = 3, and >75% = 4. The final score was determined by multiplying the intensity and the extent positivity scores, which yielded a range from 0 to 12. The mean score from each individual was calculated in tumor cells. The positive expression for markers was scored as follows: >1, IGFBP5 and >2, ADAMTS5. The positive expression of ADAMTS5 and IGFBP5 was found mainly on the cytoplasm of cells. They were presented as a brown granular material.