Primary antibody specific for cleaved caspase 3

The immunofluorescence staining protocol was adapted from O’Rourke et al⁴¹ and modified for staining and imaging in a 96-well plate. Enteroids were grown and treated in 96-well plates and on the day of staining media was replaced with 80 μL 4% paraformaldehyde and fixed for 20 minutes at room temperature. Enteroids then were washed with IF buffer and permeabilized for 20 minutes at room temperature with 80 μL 0.5% Triton X-100 (Thermo Fisher Scientific) in PBS. By this point, the Matrigel was dissolved and the enteroids remained attached to the bottom of the well. After washing with IF buffer, the enteroids were blocked for 30 minutes at room temperature with 5% BSA in PBS. The primary antibody specific for cleaved caspase-3 (Cell Signaling) was diluted 1:100 in blocking solution and incubated overnight at 4°C. After washing with IF buffer, secondary antibody specific for rabbit IgG was diluted 1:1000 in blocking buffer for 1 hour at room temperature, protected from light. Secondary antibody was removed and 4′,6-diamidino-2-phenylindole diluted 1:1000 in PBS was added for 10 minutes protected from light. The enteroids then were washed with IF buffer and then with PBS. PBS was left in the well during imaging on a Lionheart FX Automated Microscope (BioTek, Winooski, VT).