Apoptag red in situ apoptosis kit

Embryos were anaesthetized in Tricaine and fixed in 4% PFA in PBS at 4°C overnight. After fixation they were briefly washed in water then incubated in pre-chilled acetone at -20°C for 7 minutes. They were then washed in PBS + 0.1% Tween 20 (PBT) and digested with 10 µg/ml Proteinase K (Sigma) in PBT. Samples were then washed with PBT and briefly fixed in 4% PFA in PBS at room temperature. TUNEL staining was performed using the ApopTag Red in situ apoptosis kit (Millipore). Following further PBT washes, samples were incubated in equilibration buffer for 1 hour at room temperature. Samples were then incubated in reaction buffer and TdT enzyme mix at 37°C for 90 minutes. This was then replaced with stop buffer and samples incubated at 37°C for 3 hours. They were then washed in PBT and incubated with a rhodamine-labeled anti-DIG antibody diluted in blocking solution overnight at 4°C. The samples were washed thoroughly in PBT, fixed in 4% PFA in PBS for 30 minutes and washed with PBT once more. They were moved through a series of glycerol dilutions to 75% glycerol/25% PBS and mounted for confocal microscopy. Samples were imaged using an Olympus Fluoview FV1000 confocal microscope.