Histological Evaluation of Colonic Tissues

The colon of the rat was cut into distal, medial, and proximal sections, and 1 cm of different sections was fixed in 4% paraformaldehyde overnight at 4°C and then embedded in paraffin, and 4 μm thick sections were prepared. For histological analysis, sections were stained with hematoxylin/eosin (HE) and analyzed. The severity of colon damage was semiquantitatively scored according to a histological scoring scale previously described [27 (link)]. For double-label immunofluorescence immunohistochemistry, slides were blocked with 10% NDS containing 0.3% Triton X-100 at 4°C for 2 h and incubated with rabbit anti-mouse CgA (1 : 500, Abcam) in combination with a rat anti-mouse 5-HT (1 : 50; Abcam) antibody at 20°C. After washing, the sections were incubated with a mixture of Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 594-conjugated donkey anti-rat IgG (Invitrogen, Rockford, IL, USA; 1 : 1,000) secondary antibodies at 20°C for 3 h while protected from light. The sections were then mounted on glass slides and finally embedded with Fluoromount/Plus (K048, Diagonostic Biosystems, Pleasanton, CA, USA) after drying at room temperature for 30 min.