Spatial Visualization of Apoptosis Using a Whole-Mount In Situ DNA End-Labeling Technique

Apoptosis is the process by which cells activate a specific program that results in the cell’s destruction (1). Many apoptosis detection protocols take advantage of unique cellular events during this progression, the most frequent of which is endonuclease activation and the subsequent DNA cleavage into endonucleosome-sized fragments. The oligonucleosomes can be visualized as either a multimeric DNA ladder after electrophoresis of isolated genomic DNA or on fixed cells or tissue sections by labeling the 3′-OH DNA ends with a hapten-linked nucleotide using the enzyme terminal deoxynucleotidyl transferase (TdT) in the TdT-mediated dUTP nick end-labeling (TUNEL) technique (2). The spatial distribution of apoptosis within a tissue or embryo can be as crucial to interpretation as quantitation. However, current protocols do not lend themselves to precise identification of small, localized apoptotic populations within a three-dimensional (3-D) context, such as that which occurs during embryogenesis. The regions of interest are frequently too small for surgical excision and DNA extraction, and TUNEL-labeled tissue sections do not always provide clear spatial representation of apoptosis without laborious or computerized reconstruction. Vital dyes (e.g., acridine orange [AcrOr], neutral red, nile blue sulfate) are frequently used to detect programmed cell death within whole tissues (4); however, this technique is not specific for apoptosis. Moreover, the vital dye signal is transient and usually is not retained upon fixation or embedding. We have modified a commercial protocol for in situ DNA end-labeling (ApopTag; Oncor, Gaithersburg, MD, USA; Reference 5) and combined it with established protocols for the nonisotopic detection of nucleic acids (9). This protocol allows for the 3-D placement of single apoptotic cells within the much larger context of a tissue or embryo. Whole embryos or tissues no thicker than 3–4 mm are fixed in 1% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at 4°C; for larger tissues, the fixation time must be determined empirically. Hollow regions (e.g., forebrain, heart) are pierced with a needle to eliminate solvent trapping. Tissues are washed briefly in PBS (10 min at 4°C) and then dehydrated through a graded ethanol/PBS series to make them permeable and improve access for TdT and antibodies; tissues that cannot be immediately analyzed should be stored in 100% ethanol at -20°C. DNA ends are visualized using a modification of the manufacturer’s procedures for the ApopTag In Situ Cell Death Kit (Oncor; Reference 5). Embryos or tissues are rehydrated through ethanol/PBS into PBS and then incubated in equilibration buffer (Catalog No. S7110-1; Oncor) for 5 min at room temperature in sufficient volume to cover the tissue (4–5 stage-13 chick embryos at 5-μL volume each required 100 μL total). The solution is removed and replaced with working strength TdT enzyme solution for 2 h at 37°C in a humidified chamber (Catalog Nos. S7110-2 and S7110-3; Oncor; 150 μL per 4–5 embryos). TdT adds a digoxigenin-conjugated dUTP to the 3′-OH ends of the nuclear DNA fragments that are characteristic of early-stage apoptosis (2). Incubation in stop wash buffer (Catalog No. S7110-4; Oncor) for 40 min at 37°C, at 200 μL per 4–5 embryos, terminates the reaction. To detect the digoxigenin-labeled DNA ends, a modification of the whole-mount in situ hybridization protocol is used (9). Endogenous alkaline phosphatase is inactivated by incubating the tissue twice for 5 min each in Tris-buffered saline (TBST: 0.14 M NaCl, 10 mM KCl, 25 mM Tris-HCl, pH 7.0, 0.1% Tween 20) and 1 mM levamisol, followed by three washes for 5 min each in Tris-magnesium buffer (NTMT: 0.1 M NaCl, 0.1 M Tris-HCl, pH 9.5, 50 mM MgCl2, 0.1% Tween 20) and 2 mM levamisol. Embryos or tissues are then pre-blocked in TBST containing 10% sheep serum, followed by incubation for 40 min in alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer Mannheim, Indianapolis, IN, USA) diluted in NTMT according to the manufacturer’s specifications. To remove nonspecific reactivity, the antibody is pre-absorbed with acetone-extracted powder from the tissue being studied, according to established protocol (9). Following extensive washing in TBST, then NTMT (three times for 10 min for each buffer), the bound alkaline phosphatase is visualized after incubation of the tissue in 0.34 mg/mL p-nitrotetrazolium blue chloride and 0.18 mg/mL 5-bromo-4chloro-3-indolyl-phosphate in NTMT.