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The TUNEL detection principle and experimental summary of cell apoptosis

Source: Elabscience®Published: Jan 16,2024

1.Experimental principle of TUNEL method

During cell apoptosis, chromosome DNA double or single strand breaks to produce a large number of sticky 3'-OH terminus, which can be labeled to the 3' -terminus of DNA by deoxyribonucleotide terminal transferase (TdT) and derivatives formed by fluorescein, peroxidase, alkaline phosphatase or biotin. Thus, apoptotic cells can be detected by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Since normal or proliferating cells have few breaks in DNA, no 3'-OH is formed and can rarely be stained. As a result, TUNEL has become the most common method for detecting DNA fragmentation (apoptosis). (Taking TUNEL cell apoptosis detection kit (One-step TUNEL In Situ Apoptosis Kit, FITC) as an example)

Hela cells were treated with 20 U/mL Dnase for 10 minutes.


2.Key steps in TUNEL experiment

(1) Fully dewaxing and hydrating. Before dewaxing, the slices can be roasted at 60ºC for 20 min, and then dewaxed with xylene twice for 5~10 min each time. It is generally recommended to use gradient ethanol to soak from high concentration to low concentration for hydration, so that the reagent can be fully and evenly combined in the later step.

(2) Control the time of cell permeabilization. Generally, the incubation time of protease k is selected according to the thickness of the section, which is usually 10~30 min. And a few μm thick sections are used for a short time. Tens of μm thick sections are used for a long time. Conditions are reached by pre-experiment exploring so that the experimental process does not come off, but also enables the enzymes to enter the cell.

(3) Appropriately extend the incubation time of TUNEL reaction solution. It is generally incubated at 37ºC for 1 h, or a longer time can be selected according to the degree of apoptotic damage of cells. It can up to 2 h. The time should be determined by the final background coloring of the experiment.

(4) If DAB is used for chromogenic reaction, the DAB reaction generally does not exceed 10 minutes, and the background color can be controlled in combination with microscope observation, and the chromogenic reaction can be react in tens of seconds.

(5) Adequate cleaning by PBS. Cleaning after TUNEL reaction and after enzymogram reaction should strictly follow the cleaning conditions, and the number of times can be increased to 5 times to reduce the non-specific staining of the section.

(6) Blocking of endogenous POD. For tissues with large blood cell such as liver and kidney, the blocking time can be extended and the concentration of hydrogen peroxide can be increased appropriately to achieve a better blocking effect without affecting the final specific staining.


3. Timing of cell permeability

(1)The purpose of using protease K is to permeate the cell membrane and nuclear membrane, so that the reaction reagent can fully enter the nucleus for reaction and improve the positive rate. However, overhigh concentration or overlong incubation time is easy to cause tissue detachment. And the effect of protease K is similar to that of cell permeabilization reagent such as Triton X100.

(2)The general working solution concentration of protease K is 20 μg/mL, and the concentrated solution can be prepared 1~10 mg/mL when needed. Protease K can be prepared with PBS or a protease K buffer (100 mM Tris HCl pH8.0, 50 mM EDTA) and stored at -20ºC after subpackaging.

(3)The reaction time of protease K is generally 10~30 min, and the specific time is related to the thickness of the section. The film about 4 μm can be permeated for 10 min, and the section thickness about 30 μm can be extended to 30 min. If the permeability time is too long, the tissue or cells may take off from the slice. And if it is too short, the permeability effect cannot be achieved.


4. Key reagent operating conditions

(1)DAB chromogenic reaction takes about 30s~5min, and the reaction time should be controlled well and do not make the background color too dark. It may still need to explore the conditions according to the source, the thickness of the section tissue and the kit instructions.

(2)The processing time and temperature of the working solution of protease K must be found within the range. If the temperature is too high and the time is too long, it is easy to cause tissue detachment and may destroy the nucleic acid structure, resulting in false positivity.

(3)DNase I treatment of positive samples is generally recommended at room temperature for 10 minutes.


5. How to eliminate nonspecific staining

The liver or kidney, which is rich in endogenous peroxidase, requires increased hydrogen peroxide concentration and longer incubation time. In addition, the nonspecific staining can also be eliminate by enhancing inactivation, shorten the incubation time of DAB, cleaning adequately by PBS, and control the time of cell permeability appropriately under the microscope.


6. Analysis of common problems

Symptoms

Causes

Comments

Non-specific staining

The concentration of TdT enzyme is too high.

Use TdT Equilibration Buffer to dilute 1:2~1:10

The time of TdT enzyme reaction is too long or the reaction solution leaks during the TdT enzyme reaction, and the cell or tissue surface cannot be kept moist.

Pay attention to control the reaction time and ensure that the TdT enzyme reaction solution can cover the sample well.

Ultraviolet light will cause the embedding reagent to polymerize (for example, methacrylic acid will cause the fragmentation of the DNA).

Try to use other embedding materials or other polymerization reagents.

The DNA of the sample is broken when the tissue is fixed (the effect of endogenous nuclease).

Ensure that the sample is fixed immediately after sampling or fixed by hepatic vein perfusion.

Inappropriate fixatives are used, such as acidic fixatives.

Use recommended Fixative Buffer.

Some nuclease activity is still high after fixation, causing DNA breakage.

Block with a solution containing dUTP and dAPT.

Little or poor staining

Samples fixed with ethanol or methanol (the chromatin failed to cross-link with the protein during fixation, and was lost during the operation).

Fix with 4% paraformaldehyde or formalin or glutaraldehyde dissolved in PBS pH7.4.

Fixing time is too long, resulting in too high degree of cross-linking.

Reduce fixation time, or fix with 2% paraformaldehyde dissolved in PBS pH7.4.

Fluorescence quenched.

Pay attention to avoid light operation.

The permeation promotion conditions are so poor that the reagent cannot reach the target molecule or the concentration is too low.

1. Increase the reaction time of permeabilizing agent.
2. Increase the temperature of the penetrating agent (37°C).
3. Optimize the concentration and duration of proteinase K.
4. Add 0.1M sodium citrate and treat at 70°C for 30 min.

High background

Mycoplasma contamination.

Use mycoplasma stain detection kit to detect whether it is mycoplasma contamination.

The concentration of TdT enzyme is too high or the reaction time is too long.

Use TdT Equilibration Buffer to dilute 1:2~1:10 or pay attention to control the reaction time.

The autofluorescence caused by hemoglobin in red blood cells cause serious interference.
Cells that divide and proliferate at a rapid rate sometimes also have DNA breaks in the nucleus.

Other apoptosis detection kits can be selected.

Positive control has no signal

The concentration of DNase I working solution is too low.

Increase the concentration of DNase I working solution.

Loss of sample from the slides

The sample is digested by the enzyme from the slide.

Reduce the processing time of proteinase K.