Axio imager 2 system

The mice colon tissue and LS174T cell bacteria co-culture smears were fixed in 4% paraformaldehyde at 4°C for 20 min, followed by a triple washing step with PBS. The presence of Mucin 2 glycoprotein was investigated using immunocytochemistry. Briefly, fixed cultures were permeabilized with 1% Triton X-100 blocking buffer (30 min) and primary antibody anti-mucin 2 mouse mAb (Servicebio Technology Co. Ltd., Wuhan, China) at 4°C overnight. Cy3-conjugated Goat Anti-mouse antibody (Servicebio Technology Co. Ltd.) was added for 2 h at 4°C, followed by nuclear counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) for 1 min at room temperature. Limosilactobacillus mucosae ribosomal RNA (rRNA) fluorescence in situ hybridization (FISH) was performed using the EUB338 16S rRNA gene probe labeled with the 5′-Cy5 fluorophore label, and the sequence used was 5′-GCTGCCTCCCGTAGGAGT-3′ (29 (link)). Samples were observed using a Zeiss Axio Imager 2 system (Carl Zeiss, Jena, Germany).

The fixed ileum and colon tissue samples were successively dehydrated by the alcohol gradient and they were made transparent with xylene and were embedded in paraffin, and the wax blocks were cooled by a freezing table at −20 °C; then, they were sliced and preserved at room temperature. The intestinal slices were stained with hematoxylin for 7 min; differentiated with a 1% hydrochloric acid–alcohol solution for 5 s; repeatedly washed with distilled water for 15 min; stained with eosin again for 1 min; washed repeatedly with distilled water for 6 min; dehydrated with ethanol, anhydrous ethanol and xylene; covered with glass slides; and dried overnight. The PAS staining was performed according to the following instruction: after routine dewaxing, add 3% acetic acid and incubate at room temperature for 3 min; after removing the acetic acid, add the PAS solution and incubate at 37 °C for 15 min; rinse with 3% acetic acid solution for 10 s; wash repeatedly with distilled water for 4 min; dye with the nuclear fast red solution for another 5 min; wash for 4 min repeatedly with distilled water; and lastly, dehydrate and seal tablets and dry overnight. The villus height, crypt depth and the number of goblet cells were measured by the Zeiss Axio Imager 2 system (Carl Zeiss, Jena, Germany) and at least 10 visual fields were selected for statistics.