Immunohistochemical Analysis of Notch Signaling in Kaiso Transgenic Mouse Intestines

Kaiso^Tg intestinal tissues were formalin-fixed and paraffin embedded as previously described [19 (link)]. Periodic acid-Schiff (PAS) staining was performed by the John Mayberry Histology Facility at McMaster University. Immunohistochemistry (IHC) analysis of all other protein targets was performed as previously described [19 (link)], with the following modifications: antigen retrieval for chromogranin A was accomplished by heating tissues in 10 mM sodium citrate, pH 6.0 for 10 min at sub-boiling temperature; retrieval for NICD, Dll-1, Dll-4, Hes1, and Hes5 was accomplished by heating tissues at sub-boiling temperature for 15 min in TE-Tween (Tris EDTA, 0.05% Tween), pH 9.0; and retrieval for lysozyme was performed with 200 μg/mL proteinase K, 50 mM Tris pH 7.4 at RT for 5 min. Tissues were incubated with the following primary antibodies overnight at 4 °C at the indicated dilutions: rabbit anti-lysozyme (Thermo Scientific cat. #PA1–29680; 1:50); rabbit anti-chromogranin A (Abcam cat. #ab15160; 1:500); rabbit anti-Cleaved Notch 1 Val-1744 (Cell Signaling Technology cat. #4147; 1:75); rabbit anti-Hes1 (Cell Signaling Technology cat. #11988S; 1:80); rabbit anti-Hes5 (Abcam cat. #ab65077; 1:125); rabbit anti-Dll-1 (Abcam cat. #ab84620; 1:100); and goat anti-Dll-4 (R&D Systems cat. #AF1389).