Qualitative detection of apoptosis at the single-cell level by light microscopy.

• Sensitive: The maximum intensity of labeling (cell staining) of apoptotic cells is higher than the nick translation method
• Fast: The use of fluorescein-dUTP allows analysis of the samples directly after the TUNEL reaction, but before the addition of the secondary detection system
• Convenient: The direct labeling procedure using fluorescein-dUTP allows verification of the efficiency of the TUNEL reaction during the assay procedure
• Accurate: Identification of apoptosis at a molecular level (DNA-strand breaks) and identification of cells at the very early stages of apoptosis
• Flexible: No substrate included; provides the opportunity to select the staining procedure of choice

Sample material: Cytospin and cell smear preparations, adherent cells grown on slides, and frozen and paraffin-embedded tissue sections.

Widely used methods to determine apoptosis include the analysis of the genomic DNA by agarose-gel electrophoresis and DNA fragmentation assays based on ³H-thymidine and, alternatively, 5-Bromo-2'-deoxy-uridine. The methods involve the separation of fragmented, low molecular-weight DNA from unfragmented, high molecular-weight DNA in a given cell population. Thus, these methods do not provide information about the fate of an individual cell in a given cell population or, particularly, in tissue sections. Alternatively, individual apoptotic cells may be microscopically recognized because of the characteristic appearance of nuclear chromatin condensation and fragmentation, but this method is subjective and limited to a relatively narrow time window when the morphological changes are at a maximum.
The hallmark of apoptosis is DNA degradation, which in early stages is selective to the internucleosomal DNA linker regions. The DNA cleavage may yield double-stranded and single-stranded DNA breaks (nicks). Both types of breaks can be detected by labeling the free 3'-OH termini with modified nucleotides (e.g., biotin-dUTP, DIG-dUTP, fluorescein-dUTP) in an enzymatic reaction. The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes the template-independent polymerization of deoxyribonucleotides to the 3'-end of single- and double-stranded DNA. This method has also been termed TUNEL (TdT-mediated dUTP-X nick end labeling). Alternatively, free 3'-OH groups may be labeled using DNA polymerases by the template-dependent mechanism called nick translation. However, the TUNEL method is considered to be more sensitive and faster.

1. Enzyme Solution (TdT), 5 vials
2. Label Solution (fluorescein-dUTP), 5 vials
3. Converter POD (anti-fluorescein antibody-POD), ready-to-use

The In Situ Cell Death Detection Kit, POD is based on the detection of single- and double-stranded DNA breaks that occur at the early stages of apoptosis.
Apoptotic cells are fixed and permeabilized. Subsequently, the cells are incubated with the TUNEL reaction mixture that contains TdT and fluorescein-dUTP. During this incubation period, TdT catalyzes the addition of fluorescein-dUTP at free 3'-OH groups in single- and double-stranded DNA. After washing, the label incorporated at the damaged sites of the DNA is marked by an anti-fluorescein antibody conjugated with the reporter enzyme peroxidase. After washing to remove unbound enzyme conjugate, the POD retained in the immune complex is visualized by a substrate reaction.

Figure 1:Test principle.