During apoptosis, activated endonucleases cleave genomic DNA between nucleosomes. The commonly used TUNEL assay (TdT-mediated dUTP Nick End Labeling) detects DNA fragmentation in nuclei during the late stages of apoptosis.
The principle involves labeling the 3′-hydroxyl ends of fragmented DNA with fluorescently tagged dUTP, catalyzed by terminal deoxynucleotidyl transferase (TdT), allowing visualization under a microscope.
Q1: What are the common detection methods in TUNEL assays?
There are two common methods for TUNEL detection (see table below):
The first method uses TdT to incorporate fluorescently labeled dUTP into DNA breaks at the 3′-OH ends, and fluorescence is directly observed using a fluorescence or confocal microscope (e.g., with FITC labeling).
The second method involves incorporation of biotin- or digoxigenin-labeled dUTP by TdT, followed by detection with HRP-conjugated streptavidin or anti-digoxigenin antibodies. A chromogenic substrate (e.g., DAB) generates a brown precipitate that is observable under a light microscope.
|
Detection Method |
Label |
Detection Equipment |
Sample Type |
Characteristics |
|
Fluorescence |
Fluorescein-dUTP |
Fluorescence microscope |
Tissue sections, cell samples |
High sensitivity; light-sensitive |
|
Chromogenic |
Biotin/Digoxigenin-dUTP + DAB |
Light microscope |
Tissue sections |
Stable signal; requires endogenous peroxidase blocking with 3% H₂O₂ |
Q2: Why is there no positive signal in the TUNEL assay?
Lack of positive signals may result from degraded DNA in the sample, inactivated TdT enzyme in the detection reagent, degraded fluorescent dUTP, insufficient permeabilization, or excessive washing.
Recommendations:
- Include a positive control (e.g., DNase I-treated sample) to verify sample integrity and assay functionality.
- Confirm reagent validity and avoid using expired products.
- Optimize Proteinase K concentration (typically 10–20 μg/mL) and incubate for 15–30 minutes at room temperature.
- Reduce the number and duration of washes; do not use a shaker during washing steps.
Q3: Why is there nonspecific staining outside the nucleus?
Nonspecific staining in non-apoptotic regions (not overlapping with nuclear staining) may result from:
- Random DNA fragmentation in necrotic cells,
- Tissue autolysis,
- Excessive TdT or fluorescent-dUTP concentrations, or prolonged reaction times.
Mitigation strategies:
- Differentiate between apoptosis and necrosis by combining TUNEL with morphological methods such as H&E staining to identify nuclear condensation and apoptotic bodies.
- Minimize processing time and fix fresh tissues promptly.
- Lower concentrations of TdT and labeled dUTP, or shorten reaction time to reduce nonspecific signals.
Q4: Why is there high background in fluorescence detection? How can it be improved?
Common causes of high background include:
- Weak positive signals requiring strong exposure to detect,
- Autofluorescence from hemoglobin in red blood cells (in tissue samples) or contamination with mycoplasma (in cell samples),
- Inadequate washing.
Solutions:
- Include a DNase I-treated positive control to identify whether the issue is sample- or system-related.
- For autofluorescence, check blank tissue sections under the fluorescence channel. If autofluorescence is present, use quenching agents or select fluorophores that do not overlap with the autofluorescence spectrum.
- In case of mycoplasma contamination, look for irregular or punctate extracellular fluorescence and perform detection/removal accordingly.
- Improve washing by using PBS with 0.05% Tween 20 to reduce background fluorescence.
Q5: How does tissue morphology damage affect TUNEL assay results?
Excessive fixation can lead to tissue fragility and abnormal staining. It is recommended to fix for no more than 24 hours. Overdigestion with Proteinase K can damage cell structures, also resulting in abnormal staining patterns.
Q6: Can TUNEL staining be combined with immunofluorescence? If so, what is the recommended order?
Yes, TUNEL staining can be combined with immunofluorescence. It is recommended to perform TUNEL staining first, followed by immunofluorescence.
Q7: Can stained cells and tissue samples be mounted? How long can they be preserved?
Fluorescent signals in stained cell samples typically last for 1–2 days. Tissue sections can be mounted with neutral balsam, and fluorescence may remain detectable for several days to weeks. Chromogenic signals can be preserved even longer.
Q8: How should the results be analyzed after staining?
Apoptosis is analyzed by comparing the percentage of TUNEL-positive cells between groups.
Apoptotic rate = TUNEL-positive cells / total cells (DAPI or PI-stained).
Figure 1: Sobetirome reduces bleomycin-induced apoptosis in small airway epithelial cells (SAECs)
[Note]: TUNEL staining was performed on SAECs from (A) SV group, (B) SS group, (C) BV group, and (D) BS group. Blue indicates nuclei stained by DAPI, green indicates apoptotic cells positive for TUNEL staining. Scale bar: 100 µm. Arrows indicate TUNEL-positive cells. (E) Quantification of TUNEL-positive cell proportion.
That concludes the common issues encountered in TUNEL staining. We hope this guide helps junior researchers resolve similar problems effectively. Using the right tools and methods can make your work much more efficient. Wishing all researchers great success and beautiful staining results!
References
1.Li Z, Zhang X, Xie S, et al. H3K36me2 methyltransferase NSD2 orchestrates epigenetic reprogramming during spermatogenesis [published online ahead of print, 2022 Jun 23]. Nucleic Acids Res. 2022;50(12):6786–6800. doi:10.1093/nar/gkac533 (IF: 16.971)
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