Apoptosis, or programmed cell death, plays a fundamental role in many biological processes such as morphogenesis and negative selection in the immune system, as well as in multiple disease states such as cancer and neurodegenerative diseases. Apoptotic cells can be recognized by a characteristic pattern of morphological, biochemical and molecular changes.
Starting materials include: formalin fixed (wet tissue), frozen tissue in OCT, paraffin block, or slide.
Morphological changes:
Loss of cell surface structures- Cell shrinkage and shape change
- Condensation of cytoplasm and nuclei
- Nuclear envelope changes
- Nuclear fragmentation
- Apoptotic body formation
Biochemical changes:
Free calcium ion rise- bcl2/BAX interaction
- Cell dehydration
- Loss of mitochondrial membrane potential
- Proteolysis
- Phosphatidylserine externalization
- DNA denaturation
- 50-300kb endonucleolytic cleavage of chromatin
- Protein cross-linking
There are many ways of detecting apoptosis at different stages on histological sections. The most commonly used method is called TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling). One of the characteristics of apoptosis is the degradation of DNA after the activation of Ca/Mg dependent endonucleases. This DNA cleavage leads to strand breaks within the DNA.
The TUNEL method identifies apoptotic cells in situ by using terminal deoxynucleotidyl transferase (TdT) to transfer biotin-dUTP to these strand breaks of cleaved DNA. The biotin-labeled cleavage sites are then detected by reaction with HRP conjugated streptavidin and visualized by DAB showing brown color. Recently, more methods for the detection of apoptotic cells and specific parts of the apoptotic pathway are also available such as the detection of caspase activity (specifically caspase-3), fas-ligand and annexin V, etc. It is also important to use TUNEL in conjunction with other methods especially morphology, immunohistochemistry and electron microscopy.