Edta antigen retrieval solution

For immunohistochemistry, heat-induced epitope retrieval was performed by Citrate Antigen Retrieval solution (#C1031, Beijing Solarbio Science & Technology Co., Ltd.) for 40 min for vimentin, or by EDTA Antigen Retrieval solution (#ZLI-9079, ZSGB-Bio) for E-cadherin. The tissue preparations were incubated with the primary antibodies for E-cadherin (#20874-1-AP, ProteinTech Group, Inc.,) at a 1:500 dilution, vimentin (#HPA001762, Sigma-Aldrich; Merck KGaA) at a 1:250 dilution at 4°C overnight, followed by incubation with the secondary antibody EnVision™+/HRP rabbit polymer (#P0448, Dako; Agilent Technologies, Inc.) at a 1:200 dilution at room temperature for 30 min. Secondary antibody detection was performed by using the SIGMAFAST™ 3,3′-diaminobenzidine tablets (DAB Peroxidase Substrate Tablet Set, #D4168, Sigma-Aldrich; Merck KGaA). The slides were counterstained with hematoxylin for 2 min following color separation by 1% acetic acid at room temperature for 30 sec. The Samples were visualized under a light microscope (Nikon, model Eclipse E400) and images were captured using a Nikon Digital Camera (ACT-1 Nikon software).

Each FFPE block was cut into 3‐μm‐thick slices for hematoxylin and eosin (H&E) and IHC staining. The histomorphology and cytomorphology of each FFPE tumor tissue were visualized by H&E staining. The tumor areas and histological structures were evaluated by a pathologist. IHC was performed on FFPE samples taken from cell lines and xenograft samples. MLH1 and PMS2 protein expression levels were assessed using a commercial kit (ZSGB‐BIO, Beijing, People's Republic of China). After paraffin sections were dewaxed and hydrated with xylene and ethanol at different concentrations, dewaxed sections were heated for approximately 20 min in pH 9.0 EDTA antigen retrieval solution (ZSGB‐BIO) in a microwave oven. After cooling, the primary and secondary antibodies in the commercial kit were applied and incubated for 70 min and 20 min, respectively, according to the manufacturer's instructions. A DAB kit (ZSGB‐BIO) was used for the final color reaction, and hematoxylin was used for counterstaining. Tumors showing loss of nuclear staining were classified as negative for protein expression. GM12878Cas9 and HCT116 cell lines were used as external positive and negative controls, respectively.