Cell Function
Analysis and Solution of Common Problems in TUNEL Detection
Source: Elabscience®Published: Jan 16,2024
Apoptosis is a kind of programmed cell death, which is an active death regulated by specific genes, and is also a normal cell physiological response. In late apoptosis, DNA is fragmented and double-strand breaks, exposing a large number of 3'-OH sticky ends. Under the affect of deoxyribonucleotide terminal transferase (TdT), derivatives formed by deoxyribonucleotide and fluorochrome, peroxidase, alkaline phosphatase or biotin are labeled to the 3' -terminal of DNA. Detection by Fluorochrome Microscopy or Flow Cytometry is called TUNEL (TdT-mediated dUTP Nick-End Labeling). Since normal or proliferating cells have almost no DNA breaks, no 3'-OH is formed and thus there are not stained. Therefore, TUNEL has become the most commonly used method for apoptosis detection.

In TUNEL experiments, abnormal staining results are often encountered, such as weak or no fluorochrome signal, non-specific staining, and too strong fluorochrome background. So what are the causes of these problems? How can we avoid such problems? This article will answer the above questions for you.
01.Weak or no fluorescent signal

Cell or tissue samples are clearly in a state of apoptosis, but the fluorescence signal is weak or no signal detected by TUNEL kit.
(1) Inappropriate fixative reagent and fixation time.
Note: If the sample is fixed with ethanol or methanol, it will result in less efficient labeling because the chromatin fails to cross-link with the protein during fixation, resulting in chromatin being lost during operation.
Answer: Fixation with 4% paraformaldehyde in PBS pH 7.4 or formalin or glutaraldehyde is recommended. The fixed time should be appropriate. Short fixation time will lead to insufficient fixation, and the fixaion time is too long will cause overhigh cross-linking process between chromatin and protein, which is not conducive to the conbination of dUTP and chromatin.
(2) Insufficient dewaxing and hydration
Answer: When dewaxing, first bake the slices at 60ºC for 30 min, and then immerse them in xylene twice for 10 min each time. When hydration, wash the slice with gradient ethanol from high to low concentration.
(3) Poor permeablilization
Answer: Determine the time to incubate protease K according to the thickness of the section, which is usually 10 to 30 min. Explore the incubation time until the enzyme and antibody can enter the cell and the tissue on the slice can not take off. If the incubation time is too short, the cell permeablilization will not complete, and the reagent will not reach the target molecule or the concentration of reagent is too low.
(4) Fluorescence quenching
Answer: Fluorescence can be severely quenched within 10 minutes of ordinary light, suggesting that the slide should avoid light when labeling and detecting. And sample observation should be as soon as possible after the experiment. If temporary storage is required, it is recommended to store it at 4ºC in dark and the storage time preferably not more than one month.
(5) Conditions for apoptosis induction are not suitable
Answer: When DNA does not break and can not detect late signals after ultraviolet or infrared induced, you can choose other detection methods to detect cell apoptosis.
02.Non-specific staining (high false positive)

Cell or tissue samples are clearly live cells that have not been treated in any way, but when detected with the TUNEL kit, non-specific staining appears, and the fluorochrome signal is as strong as the fluorochrome signal of treated group, and even stronger than the positive control group.
Reason 1 Some cells or tissues, such as smooth muscle cells or tissues, have high levels of enzyme activity of nuclease or polymerase, which can easily lead to false positive signals. Fix cells or tissues immediately and sufficiently after obtianing to stop enzymes from causing false positives.
Reason 2 Improper fixatives, such as some acidic fixatives will cause false positives. Use recommended fixing solution is suggested.
Reason 3 Cell fixation time is too long and leads to cell autolysis, resulting in false positive. It is recommended to control the fixed time.
Reason 4 The TdT enzyme reaction time is too long or the reaction solution is leaking during the TdT enzyme reaction, and the cell or tissue surface is not kept moist. Take care to control the reaction time and ensure that the TdT enzyme reaction solution covers the sample well.
Reason 5 Incubation time of working solution is too long or working solution is not cleaned enough. Control the incubation time of the working liquid, and clean the working solution adequately after incubation.
Attention: A large number of DNA fragments can also be produced in advanced stages of necrosis or in highly proliferating/metabolizing tissue cells, which will cause false positive results.
03 Strong fluorescent background

Background staining is too strong means except for the specific staining signals, there are fluorescent signals of the same color as the specific staining interfere with our experimental results.
Q1: TUNEL dyeing time is too long
Answer: It is recommended that the dyeing time be appropriate, usually incubation at 37 ºC for 60 min.
Q2: Insufficient PBS rinsing
Answer: Insufficient rinsing of PBS after reaction of enzyme or fluorescent label working solution will lead to residue. If the background is high, the number of rinses can be increased, and incomplete cleaning can lead to non-specific staining of the slice.
Q3: Exposure time is too long
Answer: Adjust the exposure conditions, first adjust the negative group to no background light, and then shoot the experimental group with the same exposure conditions.
For more information about the TUNEL experiment, please refer to the "Principles and Experience of TUNEL Detection of Apoptosis" on the official website.