2.9. Immunofluorescence Assay

Mice were transcardially perfused, as previously reported [25,26]. Their brain and colon tissues were post-fixed, cytoprotected, frozen, sectioned, and washed [19,26]. The washed section was blocked with serum, incubated with primary antibodies for BDNF (1:200, Santa Cruz Biotechnology, #SC-65513, canonical nerve growth factor), NeuN (1:200, Millipore, #MAB377, a neuronal nuclear antigen), NF-κB (1:100, Cell Signaling Technology, #3033S), Iba1 (1:200, Thermo Fisher Scientific, #PA5-27436, a microglial protein), LPS (1:200, Abcam, #ab35654, a Gram-negative bacterial endotoxin), MUC2 (1:500, Abcam, #ab272692), claudin-1 (1:200, Santa Cruz, #G0119, an intestinal tight junction protein), claudin-5 (1:200, Millipore, ABT45, a brain tight junction protein), and/or CD11c (1:200, Abcam, #ab11029, an immune cell protein) overnight, washed, and incubated with the secondary antibodies conjugated with Alexa Fluor 594 (1:200, Invitrogen) or Alexa Fluor 488 (1:200, Invitrogen) for 2 h. The section was also stained with DAPI and photographed by a confocal laser microscope.