During cell development, programmed cell death, named apoptosis, is a fundamental process that leads to a complex series of events in the cell. Each cell compartment is involved in the process and the nucleus clearly shows DNA cleavage by endogenous endonucleases. The apoptotic process is one of the principal pathways through which organisms regulate morphogenesis, tissue differentiation and proliferation. Detecting the presence of apoptotic nuclei in tissue sections is a powerful method to evaluate cell death processes. The FragEL™ DNA Fragmentation Detection Kit allows the recognition of apoptotic nuclei on tissue sections, cryo-sections and cells fixed on slides.
The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of biotin-labeled and unlabeled deoxynucleotides to the free 3´-OH groups at the ends of DNA fragments generated by the apoptotic endonucleases. Biotinylated nucleotides are then detected using a streptavidin-horseradish peroxidase conjugate and the reaction is then revealed with diaminobenzidine (DAB), H2O2 and urea. Finally, counterstaining is performed with methyl green.
The FragEL™ kit supplies the following materials: Proteinase K (2 mg/ml) for tissue permeabilization; 5X TdT equilibration buffer; TdT enzyme; TdT labeling reaction mix, containing labeled and unlabeled deoxynucleotides; Stop buffer to terminate labeling reaction; Blocking buffer (4% BSA in PBS); 50x concentrated HRP-streptavidin; DAB tablets to reveal the reaction; H2O2-Urea tablets to reveal the reaction; 0.3% Methyl green. Two control slides, with human cells treated or untreated with 0.5 µg/ml actinomycin D to induce apoptosis, to test the kit’s specificity are also provided. The kit contains reagents for 50 reactions. Some necessary materials are not provided, such as DNase I to generate positive controls, a separate solution of hydrogen peroxide and 10 mM Tris.
The deparaffinization and rehydratation are simple histological steps; the permeabilization step is critical since over-incubation can cause cell damage and interfere with obtaining good results. The inactivation of the endogenous peroxidases is also important in order to avoid non-specific signal generation. The core of the reaction, the labeling of DNA fragments, is relatively simple but requires some technical ability. The standard protocol, which it is best to follow as written, takes about 4 hours, a relatively short time for a molecular biology-based histo/cytochemical method.
I used the FragEL™ kit to detect apoptotic cells in the development of gonads and in the germ cell differentiation of several species, including fishes and reptiles. I found it effective with very low background and clear apoptotic signal. Because some skill is required, I suggest using it after training with histochemical techniques.
| Comment on this Review |
Comments? Questions? Discuss this review of FragEL-DNA-Fragmentation-Detection-Kit-Fluorescent-TdT-Enzyme-From-Calbiochem with the rest of the Biocompare community.
|
Salvatore Valiante, PhD
Department of Biological Sciences
University of Naples Federico II
Italy
more articles...
