Immunohistochemical Analysis of 3D Organoid Cultures

Immunohistochemical techniques were carried out as previously described^{24 (link),25 (link)}. Paraffin sections of 3D organotypic cultures were baked for one hour at 60 °C and deparaffinized in xylene. Sections were then rehydrated in sequential 100%, 95%, 70% ethanol and distilled water. Antigen unmasking was performed with the Retriever (Electron Microscopy Sciences) in 10 mM sodium citrate buffer, pH 6. Endogenous peroxidases were quenched in 3% hydrogen peroxide for 6 min prior to sections being incubated for 30 min with a blocking solution of 5% BSA in PBS. Sections were incubated with primary antibodies overnight at 4 °C in 5% BSA, followed by 2 h with ImmPRESS HRP reagent (Vector Laboratories). HRP activity was then detected using DAB substrate (Thermo Scientific). Sections were dehydrated in 70%, 95%, and 100% ethanol and xylene, and coverslipped with Permount (Fisher Scientific). Sections were then imaged and photographed with an Olympus BX53 light microscope (Olympus America)^{24 (link),25 (link)}.