Here we describe the substitution of fluorescently labeled ddUTP for dUTP in the TUNEL assay to allow quantification of generated fluorescence signals by epifluorescence microscopy. The capping of DNase type I 3′OH DNA ends using ddTUNEL was further combined with phosphatase treatment for detection of DNase type II 3′PO4 ends in the same sample using a second round of ddTUNEL. Levels of modified DNA bases in tissues and fixed cultured cells could be interrogated in the ddTUNEL assay with the base modification repair enzyme formamidopyrimidine DNA glycosylase. Using rat mammary gland, from days 1 and 7 of involution, we validate the methodology's ability to label apoptotic nuclei and apoptotic inclusion bodies. In addition, we examined the types of DNA damage and modification that occur in human glioblastoma, U87 cells, following exposure to reactive oxygen stressing agents, chemotherapeutic alkylating agents, and a topoisomerase I inhibitor, irinotecan.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a common method for detecting DNA fragmentation. Originally described in 1992, terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of labeled dUTP onto 3′OH DNA ends (1). As the generation of 3′OH ends is commonly found in apoptosis, the TUNEL assay has become one of the main methods for detecting apoptotic programmed cell death.
One deficiency with the classical TUNEL assay is the use of dUTP as a substrate, which results in formation of (U)n 3′OH polymers at each DNA 3′OH primary site. Formation of indeterminate length polymers renders the assay intrinsically unquantifiable (2,3). Each addition of dUTP creates a new dU 3′OH end, precluding additional rounds of TUNEL. There are a large number of methodologies that can generate 3′OH DNA ends de novo. For example, 3′OH ends are formed when BrdU incorporated into DNA is irradiated with U V light (4). Here we describe a modified TUNEL assay called ddTUNEL, in which each 3′OH end is capped with a TUNEL-negative ddU 3′H end, thus enabling multiple sequential ddTUNEL assays to be performed on the same sample.Materials and methods
All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.Preparation of fluorescein isothiocyanate–labeled gelatin phantoms
We have developed a methodology for the production of labeled tissue phantoms that allows the quantification of fluorescence in microscopy (5). Gelatin was dissolved in 1 mL 10 mM potassium phosphate, pH 7.0, to a concentration of 3%, at 45°C. A small amount (≤100 µg) of the solid fluorescein isothiocyanate (FITC) probe was added and incubated at 45°C. The gelatin conjugate was precipitated by the addition of ice-cold ethanol, washed, dried, and resuspended in hot water.Casting and fixing of standard blocks
The labeled 3% gelatin solution was dissolved in 19% gelatin, at 45°C, to arrive at a final concentration of 15%. A concentration gradient of samples of gelatin conjugate was prepared by diluting the stock in 15% gelatin. The spectrum of the samples was taken to determine the concentration of FITC-gelatin, and aliquots were transferred into silicone molds. The molds were cooled to 4°C, and the casts were fixed in 4% paraformaldehyde (PFA). The plasticized blocks were treated in exactly the same manner as any authentic fixed tissue, being dehydrated, impregnated with wax, sliced, and mounted on slides. They were then dewaxed with xylene, rehydrated, and covered with a coverslip and sealed.ddTUNEL
A TdT reaction buffer was prepared daily diluting a stock solution 1:5 TUNEL buffer (125 mM Tris-HCl, 1 M sodium cacodylate, 1.25 mg/mL BSA, pH 6.6) and a 25 mM cobalt chloride stock solution, 1:25. The sample was washed twice in this reaction buffer, and then ~50 µL reaction buffer containing 20 U/mL TdT and 250 nM labeled ddUTP were applied to each of the samples, which were then incubated in a humidified box overnight at room temperature or for 2 h at 37°C.ddTUNEL: biotinylated ddUTP and fluorescently labeled ddUTP
Biotin-16-ddUTP (Roche Diagnostics, Indianapolis, IN, USA) labels were developed using labeled avidin, which we fluorescently labeled ourselves. PromoFluor-594 and PromoFluor-425 ddUTP were obtained from PromoCell GmbH (Heidelberg, Germany).CIAP-ddTUNEL
Each sample, having previously undergone ddTUNEL, was washed and incubated with NEBuffer 3 (New England BioLabs, Ipswich, MA, USA) for 30 min and then with a ~50-µL aliquot of the same buffer containing 100 U/mL calf intestinal alkaline phosphatase (CIAP; Sigma-Aldrich) for ≥2 h; the newly generated 3′PO4→3′OH ends were ddTUNEL-labeled.CIAP-ddTUNEL positive controls
As levels of 3′PO4 were typically very low in all of the samples we investigated, we prepared positive 3′PO4 controls using authentic DNase type II (porcine spleen). We treated fixed, permeabilized, and washed U87 cells with 10 U/mL DNase type II for 30 min at 37°C in 80 mM sodium acetate buffer, 25 mM MgCl2, pH 4.6. 3′OH ends were labeled using ddTUNEL with FITC-avidin/bitotin-ddUTP, and then half of the samples were incubated with CIAP and the other half in buffer without CIAP. The samples underwent a second round of ddTUNEL with newly generated 3′PO4 →3′OH ends labeled with Texas Red-avidin/biotin-ddUTP.The Fpg-ddTUNEL assay
Following ddTUNEL and CIAP-ddTUNEL, capping all 3′OH/3′PO4 ends, samples were washed twice in 10 mM HEPES, 10 mM NaCl, 2mM EDTA, and 0.1% BSA. Then, a ~50-µL aliquot of the same buffer containing 100 U/mL formamidopyrimidine-DNA glycosylase (Fpg; USB, Cleveland, OH, USA) was applied to each of the sections, and then incubated in a humidified box for ≥2 h. Each sample was washed twice in 1× PBS (Thermo Fisher Scientific, Rockford, IL, USA), twice in NEBuffer 3, and then a ~50-µL aliquot of the same buffer containing 100 U/mL CIAP was applied to each section and incubated for ≥2 h; samples then underwent a third round of ddTUNEL.