Diaminobenzidine chromogen

The TMA were deparaffinized and followed by antigen retrieval with citrate buffer (pH 6.0). Then 3% hydrogen peroxide and 5% goat serum were used to block the endogenous peroxidases and nonspecific antigens, respectively. The primary antibody against UPF3a (1:500, proteintech) were incubated over night at 4°C. The secondary antibody was then applied to the TMA for 1 h at room temperature after washing with phosphate-buffered saline (PBS) three times. Finally, the diaminobenzidine chromogen (Beyotime, Haimen, China) was performed to detect the positive expression of the primary antibody. The TMA ultimately was counterstained with hematoxylin and cover slipped.
After excluding loss of follow-up, 151 eligible patients were ultimately involved in the current research. Immunohistochemistry analysis was conducted following the previous description. The UPF3a expression in TMA was evaluated and semiquantitatively scored based on IHC results by two independent pathologists. The immunohistochemical staining intensity of UPF3a scored as negative (0), weak (1), moderate (2) and strong (3). Besides, the percentage of positive cells scored 5% (0), 5–30% (1), 31–50% (2) and >50% (3). Expression index = % of positive cells × staining intensity. The protein expression in CRC specimens was split into the low expression group (<4) and the high expression group (≥4) for subsequently analysis.

TMA comprised 192 primary CRC tissues that were then immunohistochemically stained to determine the CDX2 expression. Core tissue biopsy samples of 2 mm in diameter were obtained from two different regions (the tumor tissue and the area surrounding the same tumor) of individual formalin-fixed, paraffin-embedded tissue specimens for each patient. TMAs were incubated in the oven at 67°C for 90 minutes, routinely deparaffinized in xylene and ethanol of descending concentrations, and treated with citrate buffer (pH 6.0) in a pressure cooker for 20 min at 98°C for antigen retrieval. Endogenous peroxidases and nonspecific antigens were blocked by applying 3% hydrogen peroxide and 5% goat serum at room temperature for 30 minutes each. The primary antibody to CDX2 was incubated overnight at 4°C (A1629; ABclonal, Woburn, MA, USA), while the secondary antibody was incubated for 30 minutes at room temperature followed by the addition of diaminobenzidine chromogen (Beyotime, Haimen, China). Finally, the samples were counterstained with hematoxylin, dehydrated, and cover-slipped.