Multiplexable Click-iT Plus TUNEL Assays for In Situ Apoptosis Detection

Visualize apoptosis in context

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The late stages of apoptosis are characterized by changes in nuclear morphology, chromatin condensation, nuclear envelope degradation, and ultimately fragmentation of cellular DNA. While DNA-binding dyes (e.g., Hoechst™ 33342 and DAPI) are typically used to monitor nuclear morphology and chromatin condensation, DNA fragmentation is routinely detected in situ with the terminal deoxynucleotidyl transferase–dUTP nick end labeling (TUNEL) assay. Here we describe the limitations of conventional TUNEL assays and introduce the Click-iT™ Plus TUNEL Assays for In Situ Apoptosis Detection. Click-iT Plus technology enables you to perform specific and sensitive TUNEL assays that can be multiplexed with other fluorescence-based cell function assays, including those that incorporate fluorescent proteins and a variety of dyes.

The TUNEL assay…then

Since its introduction in 1992, the TUNEL assay has been widely used for the in situ detection of apoptosis. The TUNEL assay is based on the incorporation of modified thymidine analogs by the enzyme terminal deoxynucleotidyl transferase (TdT) at the 3´-OH ends of fragmented DNA. The modification on the nucleotide can be as simple as a bromine atom (5-bromo-2´-deoxyuridine triphosphate or BrdUTP) or a more complex molecule such as a fluorophore or hapten (e.g., biotin-dUTP). Incorporated BrdU is typically detected with an anti-BrdU antibody, followed by a secondary detection reagent. A fluorescently modified nucleotide (e.g., fluorescein-dUTP) can be detected directly, whereas biotin is detected indirectly by the addition of a fluorescent streptavidin conjugate.

The extensive fixation and permeabilization required to give antibodies access to the incorporated BrdU can erase important physical and antigenic characteristics of the tissue being examined. Nonspecific background issues associated with the use of biotin–streptavidin detection systems make fluorescence-based TUNEL assays preferable. However, the size of the fluorophores used to modify the nucleotides can cause a reduction in incorporation rates, decreasing the sensitivity of the TUNEL assay. Additionally, the fluorophores used in most currently available TUNEL assays suffer from high rates of photobleaching, further reducing the assay sensitivity, and they often exhibit significant spectral overlap with other fluorescent dyes and proteins, limiting the ability to multiplex with other fluorescence-based probes.

And now…Click-iT Plus TUNEL Assays

The Click-iT Plus TUNEL Assays for In Situ Apoptosis Detection were developed to address the issues affecting sensitivity, photobleaching, and multiplexability. These assays use an EdUTP (a dUTP nucleotide modified with a small alkyne moiety), which is incorporated at the 3´-OH ends of fragmented DNA by the TdT enzyme. Detection is based on a highly specific click reaction, a copper-catalyzed covalent reaction between a fluorescent Alexa Fluor™ picolyl azide dye and the alkyne moiety on the EdUTP. Because of the small size of the alkyne moiety, the EdUTP nucleotide is more readily incorporated by TdT than other modified nucleotides. In addition, the small size of the Alexa Fluor picolyl azide allows easier access to the incorporated nucleotides, negating the need for harsh DNA denaturing techniques required for antibody-based detection of the nucleotide. Moreover, the Alexa Fluor picolyl azide dye serves as a brightly fluorescent and photostable detection reagent and is available in three emission colors for flexibility in multiplex assays.

The Click-iT Plus technology improves upon the first-generation Click-iT TUNEL Assays by the use of the picolyl azide combined with a copper protectant to reduce the exposure of fluorescent proteins, such as Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP), to the damaging effects of free copper. In addition, standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT Plus EdU detection reagent to gain access to the DNA; no harsh denaturants are required. The gentle reaction and detection conditions of the Click-iT Plus TUNEL Assay enable you to multiplex this assay with fluorescent proteins, labeled phalloidins, and other copper-sensitive fluorophores (Figure 1).

Figure 1. The Click-iT Plus TUNEL Assay detects DNA strand breaks. HeLa cells transduced with CellLight™ Mitochondria-RFP, BacMam 2.0 were treated with DNase I to induce TUNEL-positive DNA strand breaks. After performing the Click-iT™ Plus TUNEL Assay with Alexa Fluor™ 647 dye, filamentous actin was stained with ActinGreen™ 488 ReadyProbes™ Reagent. Clearly identified are the mitochondria (red), the filamentous actin (green), and the TUNEL signal (purple), which is localized to the nuclei.

Specific, multiplexable TUNEL assays

To demonstrate the specificity of the Click-iT Plus TUNEL Assay in a variety of cell types, four formalin-fixed, paraffin-embedded (FFPE) tissue sections were obtained. The mouse colon, heart, liver, and intestine tissue sections were deparaffinized, fixed, and permeabilized. As a substitute for the DNA nicking that occurs in late-stage apoptosis, the tissue sections were treated with DNase I. Tissue sections without DNase I treatment were used as controls. After treatment, the nicked DNA representing the apoptotic signal was detected using the Click-iT Plus TUNEL Assay with Alexa Fluor 594 dye; no significant fluorescence was detected in the control samples (Figure 2).

To demonstrate the ability to multiplex with the Click-iT Plus TUNEL Assays, we used FFPE intestine tissue sections from a transgenic mouse in which GFP expression was localized to the muscularis externa (muscular layer surrounding the intestine). The tissue sections were subjected to deparaffinization, permeabilization, and DNase I treatment. After treatment with the Click-iT Plus TUNEL Assay (Alexa Fluor 594 dye), the tissue sections were stained with Alexa Fluor 647 dye– conjugated phalloidin and Hoechst 33342 dye. Figure 3 clearly shows that all four fluorescent signals—fragmented DNA labeled with Alexa Fluor 594 dye, muscularis externa expressing GFP, nuclei labeled with Hoechst 33342 dye, and actin staining with Alexa Fluor 647 phalloidin—can be visualized in a single tissue section.

Figure 2. The Click-iT Plus TUNEL Assay detects nicked DNA in mouse tissue sections. Formalin-fixed, paraffin-embedded (FFPE) mouse tissue from colon, heart, liver, and intestine was assayed for nicked DNA, (A) with or (B) without DNase I treatment, using the Click-iT™ Plus TUNEL Assay with Alexa Fluor™ 594 dye. Very few positive signals were detected in the sections that were not treated with DNase I.

Figure 3. The Click-iT Plus TUNEL Assay can be multiplexed with a variety of other fluorescent probes. Formalin-fixed, paraffin-embedded (FFPE) tissue from a transgenic mouse expressing GFP in intestinal muscle was treated with DNase I, followed by the Click-iT™ Plus TUNEL Assay with Alexa Fluor™ 594 dye, and then stained with Hoechst™ 33342 dye and Alexa Fluor 647 Phalloidin. (A) Cell nuclei are stained by Hoechst 33342 dye (blue), (B) the GFP signal (green) is detected in the surrounding muscular layer, (C) filamentous actin is stained by Alexa Fluor 647 Phalloidin (purple), and (D) the TUNEL-positive signal resulting from DNase I treatment is clearly defined by the Click-iT Plus TUNEL Assay with Alexa Fluor 594 dye (red). The last panel (E) is the multiplexed image resulting from an overlay of the four fluorescent signals.

Choose from three fluorescent colors

The Click-iT Plus TUNEL Assays for In Situ Apoptosis Detection are available with detection reagents that fluoresce green (Alexa Fluor 488 picolyl azide), red (Alexa Fluor 594 picolyl azide), or deep red (Alexa Fluor 647 picolyl azide). These assays have been optimized and contain all the components necessary to label and detect apoptotic cells from FFPE tissue sections or adherent cells grown on coverslips. The kits include sufficient reagents for labeling 50 coverslips (18 x 18 mm) and can be configured for 50 independent TUNEL apoptosis tests.