Tissue Preparation for Immunohistochemistry

Anesthetized (using sodium pentobarbital, 80 mg/kg, intraperitoneally) adult mice were transcardially perfused with PBS before introducing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Then, the tongue was dissected and post-fixed in 4% paraformaldehyde overnight at 4 °C. The tongues were kept in 30% sucrose solution for 5–6 d. The tissue was mounted in a cryostat embedding medium and snap-frozen in powdered dry ice. The tongues were sectioned using a cryostat (thickness of 30 µm) and kept at 4 °C in PBS for storage until used for immunohistochemistry. For the myenteric plexus, the whole mount of the duodenum was prepared as described in our previous study [19 (link)].