Citrate buffer ph 6

Staining of p-mTOR (S2448) was performed, as follows: Following the embedding of the hippocampal tissues in paraffin (Shanghai Hualing Recovery Appliance Factory, Shanghai, China), sections (5 µm) from the CA3 area were transferred to slides (Leica RM 2135, Leica Microsystems GmbH, Wetzlar, Germany) and deparaffinized. The slides were then incubated with citrate buffer (pH 6; Beijing Solarbio Science & Technology Co., Ltd.) for 5 min in a microwave oven twice (with a 10 min interval between) and cooled to room temperature. The slides were then incubated with rabbit p-mTOR polyclonal antibody (Ser2448; 1:65; YP0176; ImmunoWay Biotechnology Company, Newark, DE, USA) at 4°C overnight, and then in the anti-rabbit secondary immunoglobulin (Ig)G antibody conjugated to horseradish peroxidase (PV6001; OriGene Technologies, Inc., Beijing, China) for 30 min. A 3,3′-diaminobenzidine kit (ZLI-9017; OriGene Technologies, Inc.) was used for visualization, and slides were counterstained with hematoxylin (Beijing Solarbio Science & Technology Co., Ltd.). The average integrated optical density value was obtained by analyzing five fields per slide using Image-Pro Plus software (v. 6.0; Media Cybernetics, Carlsbad, CA, USA).

For MUC2 detection, mouse colons were fixed without flushing the luminal content with Carnoy’s fixative, embedded in paraffin, and sectioned at 3 μm. Following dewaxing and rehydration, slices were incubated for 20 min at 100°C in citrate buffer pH 6 (Solarbio, China) in a steamer for antigen retrieval. Slices were blocked for 1.5 h at room temperature in 5% BSA. Staining was performed with rabbit anti-MUC2 polyclonal antibody (1:1000; 27675-1-AP, Proteintech) and incubated overnight at 4°C. The secondary antibody was detected using an FITC-conjugated goat anti-rabbit secondary antibody (1:50; SA00003-2, Proteintech) and incubated for 1 h at room temperature. DAPI was incubated at room temperature for 10 min to stain the nuclei. The coverslips were mounted with an antifade mounting medium (Solarbio, China).