The process of cell death is tightly regulated and is essential for proper development in all multi-cellular organisms. One of the best studied cell death programs, apoptosis, is characterized by membrane blebbing, nuclear and cytosolic shrinkage and chromatin condensation. In my work, I study apoptosis during early postnatal development in transgenic mice. One of the systems that I use in these experiments is the DeadEnd Colorimetric TUNEL System from Promega.
One of the hallmarks of apoptotic cell death is DNA fragmentation. The DeadEnd Colorimetric TUNEL System is a non-radioactive technique that employs the Terminal Deoxy-Transferase (TdT) enzyme, after a proteolytic treatment of the sample, to incorporate biotinylated deoxyuridine at 3’-OH ends of DNA break sites. These modified nucleotides are then amplified using avidin-conjugated peroxidase molecules and detected by using 3,3’-deoxyaminobenzidine chromogen with hydrogen peroxide, staining apoptotic nuclei dark brown.
This system enables quantification of programmed cell death (PCD) in cell populations and I have found that it works well with both paraffin-embedded and free-floating vibratome sections. I have used it numerous times for analyzing brain sections from transgenic mice and it has generated reproducible results. It is simple to use and can be used for detecting apoptosis in cells or in tissue sections. The entire process takes about 6-7 hours.
The one thing to be aware of is that the crucial step of the protocol involves a proteolytic treatment (Proteinase K) that enhances the TDT enzyme reaction. This delicate step needs to be taken seriously or else loss of tissue, high background and poor staining can result and render a whole day’s work worthless.
In short, the DeadEnd Colorimetric TUNEL System has worked well for me. It has a number of positive features that make it worth using - it is a simple, direct, reproducible and rapid system (once the proteolytic step is optimized) and can be used on both tissues and cells.
Reena Lasrado, Ph.D.
Katholieke Universiteit Leuven