Apoptosis is a form of highly organized and programmed cell death; it does not involve lysis or damage to neighboring cells. It plays a fundamental role in many biological processes such as morphogenesis and negative selection in the immune system. In addition to its importance as a biological phenomenon, defective apoptotic processes have been implicated in an extensive variety of diseases. Excessive apoptosis causes atrophy, such as in ischemic tissue damage and neurodegeneration, whereas reduced apoptosis results in cancer. Apoptotic cells can be recognized by a characteristic pattern of morphological, biochemical and molecular changes. During apoptosis, cellular endonucleases cleave nuclear DNA between nucleosomes, producing a mix of DNA fragments whose length varies in multiples of 180 to 200 bp. Degradation of nuclear DNA into nucleosomal units is one of the hallmark features of apoptotic cell death .
The detection of DNA fragmentation relies heavily on techniques involving the extraction of nuclear DNA and characterization of such oligonucleosomal ladders by gel electrophoresis. These techniques are often tedious and time-consuming. Apoptotic endonucleases not only cleave DNA between nucleosomes, but also generate free 3’-OH groups at the ends of these DNA fragments. TUNEL (Terminal deoxynucleotidyl Transferase dUTP Nick End Labeling) allows the detection of apoptosis by labelling these 3’ OH group . Utilizing this technique, EMD Millipore's FragEL™ DNA Fragmentation Detection Kits allow the in situ recognition of apoptotic DNA fragments in paraffin-embedded tissue sections, tissue cryosections, or in cell preparations fixed on slides. I used the FragEL™ kit to detect apoptotic DNA fragments in the brain sections of rats [3,4] and gerbils [5,6] subjected to cerebral ischemia and in the sciatic nerve sections of diabetic rats [7,8]. The kit includes Proteinase K (2 mg/ml), TdT equilibration buffer (5X), fluorescein-fragEL labeling reaction mix, TdT enzyme and mounting media. The researcher needs to supply the following: xylene, ethanol (70, 80, 90, 100%), Tris buffer (10 mM, pH 8), TBS (20 mM Tris, pH 7.6; 140 mM NaCl), Coplin jars, humidified chamber, glass coverslips, Parafilm®, and optionally 1 mM MgSO4 and DNaseI (for use in generating positive control).
The DNA fragment labelling on paraffin embedded tissue sections using this kit involve a very simple process and can be completed within 3.5 h. Briefly, the paraffin embedded tissue sections were deparaffinised by immersing in fresh xylene for 5 min (2 times). Subsequently slides were rehydrated by incubating graded concentration (100%, 90%, 80% and 70%) of ethanol for 5 min each. Then slides were rinsed in TBS (20 mM Tris pH 7.6, 140 mM NaCl). Subsequently, the sections were incubated with 20 ug/ml proteinase K for 20 min at the room temperature. After rinsing in TBS, tissue sections were equilibrated with the terminal deoxynucleotidyl transferase (TdT) equilibration buffer and incubated with the labeling reaction mixture in a humidification chamber for a period of 90 min at 37°C. TdT binds to exposed 3′-OH ends of DNA fragments and catalyzes the addition of fluorescein-labeled and unlabeled deoxynucleotides. After washing in TBS, sections were mounted and observed under a fluorescent microscope. The mounting media (included in the kit) contains 4,6,-DiAmidino-2-PhenylIndole (DAPI), which allows the visualization of total cell population. My personal experience says that visualization of specimens after 3-4 h of mounting increases the sharpness of DAPI signal. The mounting media also enhances and preserves fluorescein emission thereby increasing the photostability of the fluorescein signal.
I found the kit provided a very convenient and effective method to detect apoptotic DNA fragments. One should keep in mind that TUNEL positive cell is not necessarily an apoptotic cell. For absolute identification, a battery of tests identifying different characteristic patterns of morphological, biochemical and molecular changes that occur during apoptosis is required.