Anti apc antibody

Paraffin-embedded tissue sections (4 μm) of the endometrial cancer samples were processed for immunohistochemistry. First, the specimens were deparaffinized and dehydrated, and then the sections were stained with an anti-APC antibody from Abcam 1:100 dilution); anti-PD-L1 antibody from Ventana (prediluted); anti-CD3+antibody and anti-CD8⁺ antibody from Springs (1:200 dilution); and anti-MLH1antibody, anti-MSH2, anti-MSH6, and anti-PMS2 from Maixin (1:100 dilution). After washing, the sections were incubated with biotin-conjugated secondary antibodies and subsequently with streptavidin–HRP. The sections were finally visualized by incubation with a 3,3′diaminobenzidine substrate. Images were obtained with the Mantra System (PerkinElmer, Waltham, Massachusetts, United States) with identical exposure times. The integrated optical density was used to quantify the protein levels of APC, PD-L1, CD3⁺, CD8⁺, MLH1, MSH2, MSH6, and PMS2 in the tumor tissue, and this integrated optical density was calculated by staining intensity by dividing the staining area (brown stained area).

The following primary antibodies were commercially obtained: anti-acetylated-lysine antibody (Cell Signaling Technology, 9441); anti-SIRT4 antibody (Abcam, ab10140); anti-β-catenin antibody (Cell Signaling Technology, 9562); anti-HA antibody (Santa Cruz, sc-7392); anti-Myc antibody (Santa Cruz, sc-40); anti-Flag antibody (Sigma, F1804); anti-tubulin antibody (Sigma, T619); anti-phospho-S33/37/T41 β-catenin antibody (Cell Signaling Technology, 9561); anti-β-TrCP antibody (Cell Signaling Technology, 4394); anti-β-TrCP antibody (Cell Signaling Technology, 11984) and anti-dishevelled 2 antibody (Abcam, ab228804); anti-GSK3β antibody (Cell Signaling Technology, 12456); anti-APC antibody (Abcam, ab40778). FLAG peptide was purchased from Sigma–Aldrich (St. Louis, MO). Small interfering RNAs (siRNAs) against SIRT family mRNAs were purchased from Santa Cruz Biotechnology, Inc. (SIRT1 siRNA (h), sc-40986, SIRT3 siRNA (h), sc-61555, SIRT4 siRNA (h), sc-63024, SIRT5 siRNA (h), sc-63026, SIRT6 siRNA (h), sc-63028, SIRT7 siRNA (h), sc-63030). Chemicals, including NAM (A2984) and TSA (T1952), were obtained from Sigma. Wnt3a-conditioned medium was derived from stably transfected L-Wnt3a cells.