Immunofluorescence and in situ hybridization

For tissue staining in section, mice were perfused with cold 4% PFA in PBS. The proximal 10 cm of duodenum was cleaned, flushed with cold 4% PFA, and fixed in 4% PFA for 4 hours at 4°C. The tissue was cryo-protected in 30% sucrose overnight at 4°C. Samples were embedded in OCT and 8 µm sections were prepared for immunofluorescence staining. For immunohistochemistry and in situ hybridization, the samples were fixed overnight in 4% PFA, paraffin embedded, and sectioned at 5 µm for immunohistochemistry or 10 µm for in situ hybridization. For crypt area quantitation, crypts clearly above distended granuloma tissue containing visible larval worms were called as “gran” and others were called “non-gran”. Crypt area was quantitated in ImageJ. For fetal whole mount imaging, fetal intestines were fixed in 4% PFA in PBS for 3 hrs, permeabilized, and blocked for 4 hours at room temperature. Primary and secondary antibodies were incubated at 4°C overnight. Images were acquired and processed with a Leica DM5000 B or a Zeiss Axio Imager 2 and Adobe Photoshop.