TUNEL Assay Apoptosis Detection Kit

1. Experimental materials (self-prepared)
PBS buffer (1 ×, pH ~ 7.4)
0.4% Triton X-100 (PBS formulated)
0.1% Triton X-100 in PBS containing 5 mg/mL BSA
4% Paraformaldehyde (PBS formulated)
Immunohistochemical pen
Dewaxing Solvent (Paraffin Section Sample)
Reagents related to paraffin section processing
Anti-fluorescence quenching sealed tablets
ddH₂O
2. Experimental design
A. Positive control:
Positive control slides were prepared by DNase I treatment. DNase I can digest single-stranded or double-stranded DNA to expose the 3 '-OH terminus, artificially causing apoptosis. Just do it once per experiment. (Used to verify whether there are any problems with this experimental operation and kit)
B. Negative control:
A TUNEL Reaction Buffer without TdT Enzyme was used to replace TdT Enzyme with ddH2O. (Mainly to exclude non-specific staining caused by cell apoptosis, operation process and other reasons; and to adjust the shooting exposure intensity.)
C. Experimental treatment group.
The experimental group operated normally according to the instructions.
D. Experimental control group.
The experimental group operated normally according to the instructions.
3. Experimental steps:
1. Sample preparation:
(1) For adherent cells or cell smears
a. PBS wash once.
Note: If you are worried that the cells of the cell smear will not stick firmly, you can dry the sample to make the cells stick firmly.
b. Fixation: Add an appropriate amount of 4% paraformaldehyde (formulated in PBS) and fix at 4 °C for 30 min. PBS wash twice.
c. Permeability: Add an appropriate amount of 0.4% Triton X-100 (prepared in PBS), and permeate at room temperature for 20 minutes. PBS wash twice.
d. Go to step 2. TUNEL reaction.

(2) For suspended cells or cell suspensions
A. Cells were collected (3-5 × 106 cells), centrifuged at 1000 rpm for 5 min, and washed twice with PBS.  
b. Fixation: Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) to fully resuspend the cells, and fix at 4 ℃ for 30 minutes. Centrifuge at 2000 rpm for 5 min and wash twice with PBS.  
c. Permeability: Add an appropriate amount of 0.4% Triton X-100 (prepared in PBS), and permeate at room temperature for 20 minutes. Centrifuge at 2000 rpm for 5 min and wash twice with PBS.  
d. Go to step 2. TUNEL reaction.  

(3) Paraffin tissue section
A. The paraffin sections were placed on a slicing rack and baked in an oven at 60 °C for 60 minutes.  
b. Dewaxing and hydration: Section samples were sequentially placed into xylene I (10 min) → xylene II (10 min) → 100% ethanol I (5 min) → 100% ethanol II (5 min) → 95% ethanol (5 min) → 90% ethanol (5 min) → 80% ethanol (5 min) → 70% ethanol (5 min) → ddH2O rinse for 5 min and rinse twice.  
Note: Xylene is toxic and volatile, please do this in a fume hood.  
c. Use filter paper to dry the liquid around the section sample, and circle the sample outline with an immunohistochemical pen to facilitate downstream transparency and marking.  
Note: If the outline circle of immunohistochemical strokes is found to be destroyed in subsequent experimental operations, it is necessary to make up the drawing in time.  
d. Permeability: Dilute 2 mg/mL Proteinase K solution with PBS at a ratio of 1: 50 to a final concentration of 40 µ g/mL, add 100 µ L dropwise to each sample so that the solution covers the entire sample area and incubate at 37 °C for 30 min.  
Note: Proteinase K can penetrate the cell membrane and nuclear membrane, so that the staining reagent in the subsequent step can fully enter the nucleus for reaction, and improve the labeling efficiency. Too long incubation time will increase the risk of tissue sections falling off the carrier slice in subsequent washing steps, and too short incubation time may cause insufficient permeability treatment and affect labeling efficiency. In order to obtain better results, the concentration, incubation time and temperature of Proteinase K need to be optimized according to different types of tissue samples.  
e. The slice samples were immersed in 1 × PBS and rinsed three times for 5 min each time, the excess liquid was aspirated with filter paper, and the processed samples were kept moist in a wet box.  
Note: Proteinase K must be cleaned during this step, otherwise it will seriously interfere with the subsequent labeling reaction.  
f. Go to step 2. TUNEL reaction.  

(4) Frozen tissue section
A. Fixation: Take out the frozen sections and warm them to room temperature. The slice samples were immersed in 4% paraformaldehyde (PBS formulated) and fixed at room temperature for 30 min. Section samples were immersed in 1 × PBS and rinsed three times for 10 min each.  
Note: If you are worried that formaldehyde will not be cleaned clean, it will affect the final dyeing effect. After formaldehyde fixation is completed, an appropriate amount of 2 mg/mL glycine can be added to wash for 10 minutes to neutralize the residual fixative, and then wash with PBS.  
b. Use filter paper to dry the liquid around the section sample, and circle the sample outline with an immunohistochemical pen to facilitate downstream transparency and marking.  
Note: If the outline circle of immunohistochemical strokes is found to be destroyed in subsequent experimental operations, it is necessary to make up the drawing in time.  
c. Permeability: Dilute 2 mg/mL Proteinase K solution with PBS to a final concentration of 40 µ g/mL at a ratio of 1:50, add 100 µ L dropwise to each sample so that the solution covers the entire sample area and incubate at 37 ℃ for 20 min.  
Note: Proteinase K can penetrate the cell membrane and nuclear membrane, so that the staining reagent in the subsequent step can fully enter the nucleus for reaction, and improve the labeling efficiency. Too long incubation time will increase the risk of tissue sections falling off the carrier slice in subsequent washing steps, and too short incubation time may cause insufficient permeability treatment and affect labeling efficiency. In order to obtain better results, the concentration, incubation time and temperature of Proteinase K need to be optimized according to different types of tissue samples. If optimizing the concentration and incubation time of Proteinase K still cannot improve the staining effect, you can choose to immerse the sample in 1% Triton X-100 (prepared in PBS) at room temperature for 3-5 minutes; The slice samples were then immersed in 1 × PBS and rinsed three times for 5 min each.  
d. The slice samples were immersed in 1 × PBS and rinsed three times for 5 min each time, the excess liquid was aspirated with filter paper, and the processed samples were kept moist in a wet box.  

(5) Positive treatment (only positive control is subjected to this step, other samples are directly subjected to TUNEL reaction step)
A. Dilute 10 × DNase I Buffer to 1 × DNase I Buffer with ddH20 at a ratio of 1: 10 for later use.
b. Add 100 µ L of 1 × DNase I Buffer dropwise to the processed sample covering the entire sample area and equilibrate at room temperature for 5 min.
c. DNase I (2 U/μL) was diluted 1: 100 with 1 × DNase I Buffer to a final concentration of 20 U/mL of the working solution.
d. Discard the Buffer, add 100 μL of DNase I working solution at a concentration of 20 U/mL, and incubate at room temperature for 15 min.
e. Discard the DNase I working solution and wash twice with PBS.
f. Go to step 2. TUNEL reaction.
2. TUNELreaction
(1) Prepare TUNEL reaction solution (ready for use):

(2) For adherent cells, cell smears or tissue sections
A. Add 50 μL of TUNEL reaction solution to each sample so that the reaction solution evenly covers the sample. Incubate at 37 ℃ in the dark for an appropriate time (the recommended staining time for cells is 15-30min, and the recommended staining time for tissues is 1 h).  
Note: 50μL TUNEL reaction solution is suitable for smear, section or 96-well plate (other different well plates can appropriately adjust the volume of TUNEL reaction solution to cover cells). If the sample to be tested is a smear, section or in a 24-well plate, 12-well plate or 6-well plate, you can use an anti-evaporation film, or try to use a ziplock bag or other appropriate material to cut it into a round plastic sheet slightly smaller than the well. Add TUNEL reaction solution dropwise and cover the sample, which can prevent the evaporation of TUNEL reaction solution and make the TUNEL reaction solution evenly cover the sample.  
b. Discard the TUNEL reaction solution, wash it twice with PBS, and then wash it three times with 0.1% Triton X-100 (formulated in PBS containing 5 mg/mL BSA) for 5 minutes each time, so that free unreacted markers can be cleaned.  
c. (Optional) Add an appropriate amount of DAPI dye solution with a concentration of 5μg/mL to each sample, and incubate at room temperature and protect from light for 5 minutes. After completion of staining, discard the DAPI dye solution and wash it twice with PBS for 5 min each time.  
d. (Optional) Section sealing: Add 20μL of anti-fluorescence quenching sealing tablet dropwise to each sample (anti-fluorescence quenching sealing tablet may not be suitable for some dyes, and it is recommended to conduct pre-experiment to test the matching before the experiment), cover the coverslip, and gently tap the coverslip with the blunt end of tweezers to remove air bubbles to complete the sealing.
e. Aspirate the excess liquid with filter paper, add 100 μL of PBS to the sample area to keep the sample moist, and immediately observe under a fluorescence microscope.

(3) For suspended cells or cell suspensions
A. Add 50 μL of TUNEL reaction solution to each sample tube to gently resuspend cells, and incubate at 37 °C in the dark for 15-30 minutes. The cells were gently resuspended with a micropipette every 10 min.
b. Centrifuge at 2000 rpm for 5 minutes, discard the TUNEL reaction solution, and wash twice with 0.1% Triton X-100 (PBS containing 5 mg/mL BSA) for 5 minutes each time, so that free unreacted markers can be cleaned.  
c. Each sample tube was added with 100 μL of DAPI dye solution with a concentration of 5 μg/mL, and incubated at room temperature and protected from light for 5 minutes.
d. 400 μL of PBS was added to resuspend cells and immediately detected by flow cytometry or smeared and observed under a fluorescence microscope.

When stored at-20 ℃, component A should be protected from light to avoid repeated freezing and thawing.