Publication protocol
The expression of TFF1 and CC10 was determined using immunohistochemistry, following the streptavidin biotin-peroxidase complex (SABC) method. Tissues were fixed overnight in 4% paraformaldehyde at room temperature, embedded in paraffin and sliced into 4-µm-thick serial sections. Slides were deparafinized in two treatments of xylene (each 5 min) and transferred to 100% alcohol for two treatments (each 3 min), then once through 95, 70 and 50% alcohols respectively for 3 min each. The slides were then incubated in 3% hydrogen peroxide to remove endogenous oxidases for 10 min at room temperature. After rinsing twice in PBS (5 min each), a citrate buffer were used to performed antigen retrieval (95–100°C for 10 min). Normal 5% goat serum (cat. no. 5425, Cell Signaling Technology, Inc., Danvers, MA, USA) was used as a blocking reagent and the slides were incubated for 1 h at a room temperature. The remaining steps were performed using an SABC kit purchased from Wuhan Boster Biological Technology Ltd. (Wuhan, China) and a diaminobenzidine kit (cat. no. ZLI-9017), which was purchased from Beijing ZSGB-Bio Co., Ltd. (Beijing, China). Each kit was used according to the manufacturer's protocol. PBS buffer was applied to replace primary antibodies in the negative control. Rabbit anti-human CC10 (cat. no. sc-25555) and mouse anti-human TFF1 (cat. no. sc-271464) polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and used at dilutions of 1:800 and 1:100, respectively. Slides were incubated with CC10 and TFF1 antibodies for 1 h at room temperature. Positive staining was defined by fine yellow particles in the field of view using high-magnification optical microscopy. H&E staining was conducted following a previously published protocol (24).
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