Programmed cell death, or apoptosis, is an evolutionarily conserved process involved in physiological and pathological responses of multicellular organisms to a variety of stimuli. The apoptotic cell population can be negligible with respect to total cell numbers, both in vitro and in vivo, and yet can still account for significant cell or tissue loss. It has been estimated that a 25% tissue loss per day results from apoptosis in only 2-3% of the cell population. For this reason, many researcher prefer to use in situ techinques to detect apoptosis, highlighting this phenomenon at the single cell level without diluting a sample preparation from the whole cell population (such as the homogenate for WB analysis of caspase activation).
The ApopTag technology is a TUNEL-based in situ method to detect cells with DNA strand breaks. The Terminal Deoxynucleotide Transferase dUTP Nick End Label (TUNEL) allows detection of free 3’-OH termini present in the DNA of apoptotic cell by enzymatic labeling. The exogenous nucleotides are localized by direct or indirect immuno-histochemistry or fluorescence. The Chemicon ApopTag Red In Situ Apoptosis Detection Kit contains all the reagents needed to perform the TUNEL assay on many tissue sections and to visualize with single-step immunofluoresce. The “red” is actually Rhodamine, a chromophore suitable for most fluorescent microscope work.
We found that the choice of detection method to visualize digoxigenin (i.e. immuno-peroxidase vs. immuno-fluorescence) is a critical step and must be carefully considered based on your cell types. We have been happy with the ApopTag Red Kit since we typically have to optimize conditions with other kits, where with this kit we obtained great results without high background (due to the use of histochemistry or aspecific staining). The assay worked the first time we performed it and each time since! We routinely combine the TUNEL assay with standard immuno-fluorescence to detect cellular or extracellular markers of interest by performing TUNEL first, followed by the immunofluoresce experiment. In fact, the Chemicon ApopTag Red procedure is particularly fast (the only long steps being the enzymatic reaction and the incubation with the rhodamine-conjugated Ab). In addition, by reducing the reaction volumes to 25 ul drops (we work on relatively small tissue sections) we are able to use the kit for many experiments, proving to be a cost-effective decision.
The ApopTag product line provides several simple and high-quality options. One of the main advantages when using the ApopTag Red In Situ Apoptosis Detection Kit is the low background signal. This is accounted due to the fluorescence-based, TUNEL detection techinque as opposed to the standard enzymatic histochemistry technique that most kits use. This kit, though, has the typical problem of any product sold as a “complete kit”: the relative amounts of each component are calculated on a theoretical basis and almost never match the real consumption. In this specific case, for example, the enzyme solution is the limiting reagent. In conclusion, however, I recommend the Chemicon ApopTag Red In Situ Apoptosis Detection Kit after our positive experience with the TUNEL assay combined with immuno-fluorescence localization on muscle cryosections. In particular, I suggest the use of this kit to researchers working on tissue cryosections and in need of a fast and reliable approach to assess apoptosis.
Dario Coletti, PhD
University of Rome La Spaienza
Chemicon ApopTag Red In Situ Apoptosis Detection Kit
Fast and your results have a low-background.
The relative amount of reagents you get in a kit don’t match up (i.e. the enzyme solution is the limiting reagent).
The Bottom Line
I particularly recommend this kit for working on tissue cryosections.