Tissue sections were deparaffinized twice in xylene, rehydrated in a
graded ethanol series, and rinsed in distilled water. A steamer was used for
heat antigen retrieval by incubating slides in a citrate acid buffer solution
(pH 6.0) at 96 °C for 20 min. After cooling the jejunum tissue, it was
permeabilized with 0.5 % Triton X-100 for 10 min, followed by washing three
times with Hank’s balanced salt solution (HBSS). Then, the tissue was
incubated with 5 % bovine serum albumin (BSA) at 37 °C for 1 h to reduce
nonspecific background. For Ulex europaeus agglutinin 1 (UEA-1) staining,
secretory cells in the jejunum were stained with anti-UEA-1 antibodies (1:200,
Sigma, L9006, China) for 1 h, followed by 4′,6-diamidino-2-phenylindole
(DAPI) (1:5,000, Invitrogen™, D1306, China) for 5 min at room
temperature. For lysozyme (Lyz) staining, cells were stained with primary
antibodies (anti-rabbit lysozyme antibody, 1:200, Abcam, China) overnight at
4°C. The samples were incubated with goat anti-rabbit to Alexa Fluor 594
(1:250, Abcam China) for 90 min, followed by DAPI for 5 min at room temperature.
The samples were examined with a Zeiss 710 laser scanning confocal microscope.
Fluorescence images were collected for further qualitative and quantitative
analysis.
graded ethanol series, and rinsed in distilled water. A steamer was used for
heat antigen retrieval by incubating slides in a citrate acid buffer solution
(pH 6.0) at 96 °C for 20 min. After cooling the jejunum tissue, it was
permeabilized with 0.5 % Triton X-100 for 10 min, followed by washing three
times with Hank’s balanced salt solution (HBSS). Then, the tissue was
incubated with 5 % bovine serum albumin (BSA) at 37 °C for 1 h to reduce
nonspecific background. For Ulex europaeus agglutinin 1 (UEA-1) staining,
secretory cells in the jejunum were stained with anti-UEA-1 antibodies (1:200,
Sigma, L9006, China) for 1 h, followed by 4′,6-diamidino-2-phenylindole
(DAPI) (1:5,000, Invitrogen™, D1306, China) for 5 min at room
temperature. For lysozyme (Lyz) staining, cells were stained with primary
antibodies (anti-rabbit lysozyme antibody, 1:200, Abcam, China) overnight at
4°C. The samples were incubated with goat anti-rabbit to Alexa Fluor 594
(1:250, Abcam China) for 90 min, followed by DAPI for 5 min at room temperature.
The samples were examined with a Zeiss 710 laser scanning confocal microscope.
Fluorescence images were collected for further qualitative and quantitative
analysis.