Goat anti mouse jam a

Frozen murine colon sections collected on SuperFrost glass slides (Thermo Scientific, Waltham, MA, USA) were fixed in 4% (v/v) paraformaldehyde for 10 min at room temperature and permeabilized with 0.1% (v/v) Triton X-100 in PBS (all from Sigma-Aldrich) for 20 min at room temperature. The sections were blocked for 1 h with 1% (w/v) BSA, and incubated with the following primary antibodies for 1 h at room temperature: rabbit anti-mouse MUC2 (1:25; #sc-15334, Santa Cruz, Santa Cruz, CA, USA), mouse anti-mouse α-SMA (1:200; #a2547, Sigma-Aldrich), goat anti-mouse JAM-A (1:20, #AF1077, R&D) followed by 40 min of incubation at room temperature with Alexa Fluor 488-conjugated goat anti-rabbit (1:500; #A11034, Invitrogen, Waltham, MA, USA), Alexa Fluor 594-conjugated goat anti-mouse (1:500; #A11005, Invitrogen) and Alexa Fluor 488-conjugated donkey anti-goat (1:500; #A11055, Invitrogen), respectively. DAPI (1:25,000, Invitrogen) was used for nuclear staining. Coverslips were finally mounted with Fluorescence Mounting Medium (#S3023, Dako, Santa Clara, CA, USA). The images were acquired by Leica SP8 Confocal Microscopy using a 60X objective and analyzed using ImageJ.