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For immunofluorescence analysis, the sections were heated in a microwave oven in citrate buffer, after cooling, blocked with 5% BSA in PBS and 0.5% (wt/vol) Triton X-100 for 30 min at room temperature, and subsequently incubated with primary antibodies at 4 C overnight and secondary antibodies for 1 h at room temperature, followed by washing and staining with DAPI (Solarbio, S2110)

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Thus, in situ produced microbial folate may favourably influence the cellular nutrition of the local colonocytes and may be important in maintaining intestinal homeostasis and modulating gut microbiome function, e.g., through regulation of colon mucosal proliferation (i.e., colorectal cancer prevention) and its anti-inflammatory effects 248,249,256,257,277,278,279,280,281,282,283,284,285,286

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