Common Problems and Solutions in TUNEL Assay for Detecting Cell Apoptosis

Common Problems and Solutions in TUNEL Assay for Detecting Cell Apoptosis

Hello everyone, I'm your experimental assistant. In the previous issue, we discussed various methods for detecting cell apoptosis. In this issue, we will focus on the TUNEL assay for detecting cell apoptosis and analyze the reasons for the failure of TUNEL staining results in experiments. First, let's understand the steps of the TUNEL staining process, as shown in the diagram below.

Figure 1. TUNEL Staining Procedure (Note: Nuclear staining steps are optional)

When using the TUNEL assay kit to detect apoptosis in cells, various reagents such as proteinase K, fluorescein-dUTP, TdT enzyme, etc., are required. In addition, positive and negative control groups need to be set up. Faced with the complex staining steps, many people have expressed confusion. Below, we will provide answers one by one.


Main Components and Functions of the TUNEL Assay Kit

1) Equilibration Buffer: Used to maintain certain reaction conditions, with Mg2+ in the buffer reducing background and Mn2+ enhancing staining efficiency.

2) Proteinase K: Permeabilizes cell membranes and nuclear membranes to ensure the entry of TUNEL probes into cells and ensure sufficient staining.

3) Fluorescein-dUTP: Labels the 3'-OH ends and serves as a substrate for TdT enzyme.

4) TdT Enzyme: Key enzyme catalyzing the labeling of 3'-OH ends with dUTP.


Positive Control Group, Negative Control Group, Experimental Group

1) Positive Control Group: Treated with DNase enzyme to induce cell apoptosis and expose 3'-OH ends. One sample is prepared for each experiment.

2) Negative Control Group: TdT enzyme is not added, and all other steps are the same as the experimental group. One negative control is required for each type of tissue sample. For example, if there are 5 lung tissue sections from mice, then 5 negative control samples are needed.

3) Experimental Group: In addition to the positive and negative control groups, all other samples are considered experimental groups.


Why Set Up Positive and Negative Control Groups

1) Positive Control Group: Used to verify whether there are any issues with the experimental procedures and the assay kit.

2) Negative Control Group: a. To exclude nonspecific staining caused by reasons such as cell apoptosis and the experimental process itself. b. To adjust the exposure intensity during imaging.

In addition, individuals may encounter various issues with staining results, including weak fluorescence signals, no fluorescence signals, high false positives, strong fluorescence background, etc. The reasons for staining failure mainly include improper sample handling, insufficient staining, prolonged exposure time, etc. These issues can be analyzed and addressed accordingly from the following perspectives.


Weak or Absent Fluorescence Signals

If it is known that the cell or tissue samples are in a state of apoptosis, but fluorescence signals are found to be weak or absent when using the TUNEL assay kit.

4.1 Improper Sample Handling

1) The sample slices are too thick. It is recommended to slightly thin the slices for better staining.

2) Inadequate deparaffinization and hydration. It is recommended to deparaffinize at 60°C for 20 minutes and then use xylene twice for 5-10 minutes each; for hydration, use a gradient of ethanol from high to low concentrations.

3) Proteinase K incubation time is too short. Optimize the Proteinase K incubation time, commonly used for 10-30 minutes. Slices of around 4 μm are typically incubated for 10 minutes, while slices around 30 μm are incubated for 30 minutes.

4) Proteinase K concentration is too low. It is recommended to use a working concentration of Proteinase K at 20 μg/mL.

5) Slices stored at -20°C for a long time may not be fresh, reducing staining efficiency. It is recommended to use fresh slices.

4.2 Improper Staining Procedures

6) Staining time is too short. It is recommended to incubate at 37°C for 60 minutes, which can be extended up to 2 hours depending on the degree of apoptosis damage, but consider background staining.

7) TdT enzyme or fluorescence-labeled dUTP concentration is too low. It is recommended to appropriately increase the concentration of TdT enzyme or fluorescence-labeled dUTP.

8) TdT enzyme inactivation. It is recommended to prepare the TUNEL reaction solution just before use and store it briefly on ice. Prolonged storage can lead to TdT enzyme inactivation.

9) Sample drying. After adding the TUNEL reaction solution, cover the slides with cover slips/film/wet boxes to ensure uniform staining of the samples and prevent the reaction solution from drying out.

4.3 Improper Fluorescence Detection Procedures

10) Operation without avoiding light. Due to the fragile nature of fluorescence, it is recommended to avoid light when labeling and detecting samples, and observe them as quickly as possible.


Non-specific staining (high false positive rate)

If it is known that the cell or tissue samples are still in a live cell state, but non-specific staining occurs with the TUNEL assay kit, showing fluorescence signals similar in intensity to processed samples, or even stronger than the positive control group's fluorescence signals.


5.1 Improper sample processing

1) The use of acidic or alkaline fixatives may cause DNA damage. It is recommended to use a neutral pH fixative solution to fix tissues or cells.

2) Excessive concentration of fixative solution may lead to cell self-dissolution, irregular DNA strand breakage, causing false positives. It is recommended to use a 4% paraformaldehyde solution (dissolved in PBS).

3) Prolonged fixation time may lead to cell self-dissolution, irregular DNA strand breakage, causing false positives. It is recommended to control the fixation time at 4°C for 25 min.

4) Excessive treatment time with Proteinase K may disrupt nucleic acid structure, resulting in false positives. It is recommended to shorten the treatment time appropriately.

5) Excessive concentration of Proteinase K may disrupt nucleic acid structure, resulting in false positives. It is recommended to adjust the concentration of Proteinase K solution appropriately, with a general working concentration of 20 μg/mL.

5.2 Improper staining procedure

6) Excessive TUNEL staining time. It is generally recommended to incubate at 37°C for 60 min, which can be extended to 2 h depending on the degree of apoptotic damage, but should be combined with background staining.

7) Insufficient washing of samples. After staining, increase the number of PBS washes to 5 times to avoid nonspecific staining of sections.


Strong fluorescence background


6.1 Improper staining procedure

1) Excessive staining time for TUNEL. It is recommended to adjust the staining time appropriately, typically incubating at 37°C for 60 minutes to avoid background staining.

2) Excessive concentration of TUNEL staining solution. It is suggested to increase the dilution ratio to optimize the concentration.

3) Utilize the divalent cations provided in the kit to optimize the reaction. The Mg2+ in the kit can reduce background, while Mn2+ can enhance staining efficiency.

4) Insufficient washing of samples with PBS. After the TUNEL reaction, increase the number of PBS washes to 5 to remove any residual dye in the tissue sections.

6.2 Improper fluorescence detection operation

5) Prolonged exposure time. Adjust the exposure conditions by first optimizing for no background light with the negative control group. Then, use these exposure conditions to capture images of the experimental group (fine-tuning adjustments may be made, but significant adjustments are unnecessary).

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