Immunofluorescence

Mice were anesthetized and perfused with 20 ml ice-cold 0.1 mol l⁻¹ phosphate-buffered saline (PBS) via the cardiac apex, followed by perfusion with 4% paraformaldehyde (PFA; Zhuang et al., 2020). Whole brains were collected and immersed in 4% PFA overnight and 30% sucrose solutions for 72 h at 4°C. Then, the brain samples were cut into 10 μm coronal slices and fixed on slides. The colon tissues were fixed in 4% PFA overnight and cut into 5 μm thick sections. The tissue sections were washed with PBS and after the processing blocked with 5% BSA and 0.3% Triton X-100. Then, the sections were incubated overnight with the following antibodies: Iba-1 (1:500, Abcam, ab5076), CD68 (1:200, Bioss, bs-6049R), Arg-1 (1:500, Proteintech, 16001-1-AP), and Claudin (1:50, Santa Cruz Biotechnology, sc-166338) at 4°C. After washing with PBS, the cryosections were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen, A-21206), Alexa Fluor 594-conjugated donkey anti-goat IgG (Invitrogen, A-11058), Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen, A-21202), Alexa Fluor Plus 594-conjugated donkey anti-mouse IgG (Invitrogen, A-32744), Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Invitrogen, A-31572), Alexa Fluor 488-conjugated donkey anti-goat IgG (Invitrogen, A-32814) and Alexa Fluor 488-conjugated donkey anti-rat IgG (Invitrogen, A-21208) at 37°C for 1 h. Finally, the brain sections were stained with DAPI (Abcam, ab104135), and observed and analyzed under a fluorescence microscope (Leica, Mannheim, Germany). Three sections from each mouse were examined. The mean number of stained cells in each brain section was examined in three fields. The mean fluorescence intensity in each colon section was analyzed using Image J software (NIH).