TACS•XL® in situ Apoptosis Detection Kit From Trevigen

TUNEL (TdT dUTP nick end labeling) assay has become a widely accepted method to detect nuclear DNA fragmentation, which characterizes cell apoptotic processes. In brief, terminal deoxynucleotidyl transferase enzyme (TdT) incorporates labeled nucleotides onto the 3’-OH ends of DNA fragments in tissue or cell samples, allowing the detection of DNA fragmentation.

The TdT included in the kit incorporates brominated nucleotides (BrdU) more efficiently than biotinylated nucleotides which are used by other kits; this increases resolution and improves the signal/noise ratio. In addition, the kit provides a non-lipophilic detergent-based buffer (cytonin) which permits optimization of the permeabilization step, avoiding (not always) the over-permeabilization of samples that can occur when proteinase K is used.

All reagents required for the in situ reaction are provided (can be purchased separately): proteinase K and cytonin; TdT enzyme, stop and labeling buffer; Brd-dNTP mix; Streptavidin-HRP and streptavidin diluent; DAB solution and DAB enhancer; Anti-BrdU antibody; TACS nuclease and buffer; Methyl green.

Once rehydrated with PBS, tissue slides or immobilized cells are processed through the permeabilization step (with proteinase K or cytonin) and then through the endogenous peroxidases blocking step. Afterwards, slides/cells are incubated with the labeling mix for 1 hour at 37ºC; during this time, BrdU labeling occurs. The anti-BrdU antibody is then added and incubated for 30 minutes at 37ºC and then the streptavidin-HRP is added. The streptavidin binds the biotin-labeled anti-BrdU antibody. Finally, the DAB chromogen is added to react with HRP and methyl green is used to counterstain sections. The total time to complete the assay is 3.5 hours.

The kit provides a detailed datasheet containing helpful information about the whole procedure, including how to produce both positive and negative controls, a good troubleshooting guide, a complete section for double-labeling reactions, and recommendations for electron microscopy applications. The cost is lower than other commercial kits.

I have been using the TACS•XL® in situ Apoptosis Detection Kit for the detection of apoptotic nuclei in several tissues, such as brain, liver and pancreas, of non-obese diabetic mice (NOD) and have obtained very good results. I have also used it to study the apoptotic pathways of elasmobranch gametogenesis.