Ab122769

To evaluate the expression and distribution of three key genes related to tumor stemness in colorectal cancer and normal tissues, we performed multiplex immunofluorescence staining using the PANO 7-plex IHC kit (Cat. No. 0004100100; Panovue, Beijing, China) and Tyramide Signal Amplification Fluorescence Kit (Panovue, Beijing, China) (Yu et al., 2012 (link)). We established the colorectal cancer tissue microarrays (TMAs) consisting of primary tumor, metastatic tumor, and matched normal tissue from cancer patients who had been confirmed by pathological examination with hematoxylin and eosin (H&E) staining. Each of these tissues was cut into pieces of 1.0 mm and attached to the slides (5 mm thick) from the TMAs. The TMAs were incubated with anti-PARPBP (ab211634; 1:100; Abcam, Cambridge, United Kingdom), anti-KNSTRN (ab122769; 1:100; Abcam, Cambridge, United Kingdom), and anti-KIF2C (12139-1-AP; 1:200; Proteintech, Rosemont, IL, United States) antibodies at 4°C overnight, and then with horseradish peroxidase-conjugated secondary antibody and tyramide. A microwave was used to heat-regenerate the TMAs after each Tyramide Signal Amplification step. We used 4′,6-diamidino-2-phenylindole (DAPI) to counterstain the cell nuclei. The multiplex immunofluorescence image analysis is shown in Supplementary Materials.

We collected a total of 182 progesterone receptor positive human endometrial tissue samples, 107 stage III-IV cancer tissue of which had accompanying follow-up information, and 75 cancer-adjacent endometrial tissue samples from archives of paraffin-embedded tissues between May, 2011 and May, 2014 at the Department of Pathology of Peking Union Medical College Hospital. The follow-up was performed until May 30, 2019. The pathological diagnoses were reconfirmed by a pathologist. The project was approved by the Ethical Committee (Peking Union Medical College Hospital), and informed consent was acquired from patients or family members. IHC was performed as previously described (22 (link)). Anti-antibody (AKT1 1:250, Abcam, ab235958; PR 1:100, Abcam, ab32085; PBK 1:100, Abcam, ab75987; BIRC5 1:800, Abcam, ab469; AURKA 1:100, Abcam, ab52973; GTSE1 1:50, Abcam, ab103232; KNSTRN 1:50, Abcam, ab122769; PSMB10 1:100, Abcam, ab183506) was used for IHC. The scoring details have been described previously (23 (link)).