Fitc conjugated goat anti mouse secondary antibody

Segments of distal colonic tissue (1 cm) collected from the mice were clipped and fixed with 10% neutral-buffered formalin (Hepeng Biology, Shanghai, China) for 24 h. Colonic tissue samples were embedded in paraffin and cut into cross sections with a thickness of 4 μm. The cross sections were then stained with hematoxylin and eosin for histological analysis. To prepare the samples for immunofluorescence analysis, the colonic sections were deparaffinized, soaked with 0.01 M Tris/EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 10 min, and permeabilized in 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). After the sections were blocked by PBS containing 5% BSA, they were incubated with the monoclonal primary antibody for 5-HT (1:500, GeneTex, Irvine, CA, USA). Finally, the sections were treated with the FITC-conjugated goat anti-mouse secondary antibody (Beyotime, Shanghai, China) and DAPI (Life Technologies, Carlsbad, CA, USA). Immunofluorescent images were acquired by using laser scanning confocal microscopy (Leica, Wetzlar, Germany). Five fields were randomly selected from each section, and the thickness of muscle and the depth of crypts were measured using Caseviewer software [22 (link)].