Intense NTN-like immunostaining localized to the intestinal crypt and villus epithelium and muscle layers. To localize the sites of NTN protein expression, NTN immunohistochemistry was performed on 30 Ém cryo-sections of tissues fixed by Zamboni's fixative. To block endogenous peroxidase activity, sections were treated with 0.3% hydrogen peroxide in 50% ethanol for 30-60 min and washed extensively in 50% ethanol and PBS. To block non-specific binding sites, sections were treated for 90 min with TBS containing 1% bovine serum albumin (BSA) and 10% normal rabbit serum. Then sections were incubated at 4°C overnight with Sheep anti-rhNTN: whole serum (S-010-100) at 1:2000 dilution in PBS containing 0.1% Triton X-100 and 1% normal rabbit serum. After washing, the sections were incubated with a rabbit anti-sheep biotinylated IgG at 1:200. The sections were washed and incubated with ABC kit (Vector Lab) at 1:100 for 2 hours.