While a sample that undergoes ddTUNEL and then CIAP-ddTUNEL leads to capping of all 3′OH/3′PO4 ends, incubation with Fpg/CIAP, followed by ddTUNEL, labels all the newly Fpg-generated 3′OH ends derived from modified bases and AP sites.
AP sites are only hydrolyzed by Fpg if the site contains the aldehyde tautomeric form of deoxyribose (21-24). Reduction with sodium borohydride (NaBH4) (25) or incubation with DNP-H renders AP sites Fpg-negative. AP sites converted into hydrazones by DNP-H can be interrogated independently with a labeled anti-DNP antibody (Supplementary Figure S2).Quantification of ddTUNEL-labeled fluorescence signals
To quantify signals from different labeled ddUTP probes, we developed a methodology to prepare gelatin-based tissue phantoms containing known amounts of covalently bound fluorophore.
We covalently labeled gelatin with amine-reactive fluorophoric dyes that are cast into molds, fixed using paraformaldehyde, impregnated with wax, sectioned to a known thickness, mounted on slides, dewaxed, rehydrated, and then used to prepare standard curves of fluorescence versus fluorophore concentration.
By combining fluorescently labeled tissue phantoms, we were able to quantify the levels of different types of DNA damage in fixed tissue.
Figure 2A shows typical images obtained from FITC-gelatin phantoms that were then used to prepare a calibration curve (Figure 2B). The 6 µm–thick FITC-gelatin phantom images (Figure 2A; I to VII) were recorded using 40× magnification and an accumulation time of 100 ms. The final image (Figure 2A; VIII) shows background levels of autofluorescence in unconjugated gelatin and was accumulated over 10s.
The signal was proportional to concentration, with R2 = 0.99 (Figure 2B). The standard deviation of the FITC signal, at each concentration and using three different slides, averages 7.3% of signal. Figure 2C shows the relationship between signal and accumulation time, using three different 11.5-µM samples measured using 100× magnification. The relationship is linear, with R2 = 0.994 and SD = 5.3% signal mean.ddTUNEL to study mammary involution
To validate the ddTUNEL assay, we used the assay to characterize the process of mammary involution. Mammary involution is characterized by a massive loss of epithelial cells due to apoptosis, and organ sculpting in this tissue has been widely studied (7-9). The final processing of DNA digestion products, released from epithelial cells in apoptotic bodies, occurs within the macrophages/lymphocytes of the immune system (26-28). Figure 3, A–D, shows the presence of ddTUNEL-positive nuclei and the formation of apoptotic bodies at different magnifications in breast tissue on day 1; while Figure 3, E–H, shows day 7 of involution. It can be seen that the apoptotic bodies contain both ddTUNEL- and Fpg-ddTUNEL–positive DNA. Moreover, this DNA is associated with histone γ-H2A.X (Figure 3, D and H).
It should be noted that the labeled ddTUNEL probes in Figure 3, D and H were switched and compared with Figure 3, A–C and E–G, to demonstrate that the fluorescence of the apoptotic bodies is neither an artifact caused by nonspecific binding of the probes nor due to background fluorescence (28). The red/green arrow in Figure 3, C and D, shows that the apoptotic bodies have high levels of Fpg-ddTUNEL–positive DNA, and this DNA is co-localized with phosphorylated histone γ-H2A.X.
In Figure 3B, a pair of ddTUNEL-positive cells with a halo of digested oligonucleotides around denuded 4′,6-diamidino-2-phenylindole (DAPI) nuclei can be observed (arrow). In this case, biotin-ddUTP/FITC-avidin was used in the ddTUNEL assay, and the FITC-avidin had a ratio of FITC to protein of 1.03:1. The average signal correlates to an FITC phantom concentration of 24 µM, and we could therefore determine that, in this pair of cells, there was approximately one 3′OH for every 640bp. A cell nearby (dotted arrow) has almost completed its apoptotic death and has a 3′OH for every 105bp. If a cell is digested into the theoretical minimum-sized 180- to 200-bp fragments, there would be a 3′OH for every 95bp, ignoring the contribution of nicks. The average number of cuts/nicks in the cells that do not appear to be apoptotic is in the order of one free 3′OH for every 15,000bp.