Ab171972

Tissue microarray of PC patients was obtained from Shanghai Outdo Biotech Company (#JS W-11-01), which included 87 tumor tissues and 45 para-carcinoma tissues. Ethical approval was obtained from the Ethics Committee of First Affiliated Hospital, School of Medicine, Shihezi University. In brief, tumor specimens were dewaxed by xylene for 15 min and rehydrated by 100% alcohol for 10 min. The antigen was repaired in 0.01 M sodium citrate buffer (pH = 6.0) at 180 ℃ for 5 min and blocked by 3% hydrogen peroxide bath for 15 min. After washing, the sections were incubated with anti-CACYBP (1: 50, Cat. # ab171972, Abcam) or anti-Ki67 (1:200, Cat. # ab16667, Abcam) overnight at 4 ℃ and followed by conjugation to the Goat Anti-Rabbit IgG H&L (HRP) antibody (1: 400, Abcam, USA, Cat. # ab6721) at room temperature for 2 h. Tissue sections were stained with DAB and hematoxylin, and then exanimated with microscopic (Olympus). All specimens were classified into categories based on staining percentage and staining intensity as previously described (Feng et al. 2021 (link)).

Human microarray chip (Cat No. HBiDC122Su01) including CCA tissue as well as normal non-carcinoma tissue was provided by Shanghai Outdo Biotech Company. Corresponding information of patients and written informed consent were collected. This work conducted IHC analysis for detecting CACYBP levels within tumor and normal tissues. Briefly, xylene was added for tissue section dewaxing, while ethanol at different concentrations for rehydration. The antigen was repaired by citrate buffer following the blocking of 3% hydrogen peroxide solution. Subsequently, sections were then incubated using primary antibody CACYBP (1:50; Cat. #ab171972; Abcam, Cambridge, UK) together with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody (1:400; Cat. #ab97080; Abcam), followed by staining with diaminobenzene (DAB) as well as hematoxylin, observation in a microscope and evaluation independently by Haonon et al.’s method [37 (link)] via two pathologists.