TUNEL assay and immunofluorescence

Apoptosis was measured at the single-cell level in the paraffin-embedded tissue sections or in single-cell suspension using the In Situ Cell Death Detection Kit (Sigma-Aldrich) labeled with TMR red fluorescence following the manufacturer’s instructions.

The tissue sections were first deparaffinized and serially rehydrated, permeabilized at room temperature, and incubated with TUNEL mixture at 37 °C for 1 h in the dark. Nuclei were stained with DAPI and sections were mounted using ProLong Diamond Antifade Mountant (ThermoFisher). TUNEL staining was analyzed using fluorescence microscopy. Eight slides (each containing at least 1 gland) per treatment group were stained. For each gland, multiple high-power (40×) images were taken. Alexa 488-labeled TUNEL-stained cells were overlaid with DAPI-stained nuclei (representing all cells). The average number of cells undergoing apoptosis was determined by counting the number of TUNEL-positive cells in a high-power field and normalizing with the total number of DAPI-positive cells.