TUNEL apoptosis kit (red fluorescence)

1. Experimental materials (self-prepared)
PBS buffer (1 ×, pH ~ 7.4)
0.2% Triton X-100 (formulated in PBS)
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 sealing tablet
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 produce monodeoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides endonucleases, artificially causing apoptosis.
B. Negative control:
Using TUNEL Reaction Buffer without TdT Enzyme, using ddH₂O replaces TdT Enzyme.
C. Experimental treatment group.
D. Experimental control group.
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 0.2% Triton X-100 (prepared in PBS) and permeate at room temperature for 20 minutes. PBS wash twice.
d. Transfer steps2. TUNELreaction。

(2) For suspended cells or cell suspensions
a. Cells were harvested (3-5 × 10⁶Cells), 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 0.2% 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. Transfer steps2. TUNELreaction。

(3) Paraffin tissue section
A. Dewaxing and hydration: Section samples are 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 minutes and rinse twice.
Note: Xylene is toxic and volatile, please do this in a fume hood.
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 20 µ g/mL at a ratio of 1: 100, add 100 µ L dropwise to each sample so that the solution covers the entire sample area and incubate at 20-37 ℃ 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.
d. Sections were rinsed twice with PBS for 5 min each time, excess liquid was aspirated with filter paper, and 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.
e. Transfer steps2. TUNELreaction。

(4) Frozen tissue section
a. Fixation: Remove the frozen sections and warm them to room temperature. Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and fix it at room temperature for 30 minutes. PBS was rinsed twice for 10 min each time.
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 20 µ g/mL at a ratio of 1: 100, add 100 µ L dropwise to each sample so that the solution covers the entire sample area and incubate at 20-37 ℃ 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.
d. Sections were rinsed twice with PBS for 5 min each time, excess liquid was aspirated with filter paper, and 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.
e. Transfer steps2. TUNELreaction。

(5) Positive treatment (only positive control is subjected to this step, other samples are directly subjected to TUNEL reaction step)
a. With ddH at a ratio of 1: 10₂0 10 × DNase I Buffer was diluted to 1 × DNase I Buffer 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 10 min.
e. Discard the DNase I working solution and wash twice with PBS.
f. Transfer steps2. TUNELreaction。
2. TUNELreaction
(1) Prepare TUNEL reaction solution (ready for use):

50 μL

(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 ℃ for an appropriate time in the dark (the recommended staining time for cells is 30min-1h, and the recommended staining time for tissues is 2h).
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, rinse it twice with PBS, and then rinse 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, the DAPI stain solution was discarded and rinsed twice with PBS for 5 min each time.
d. (Optional) Section sealing: Add 50μL 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 a 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. Use filter paper to suck off excess liquid, add 100μL 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 the cells, and incubate at 37 °C in the dark for 30-60 minutes. The cells were gently resuspended with a micropipette every 15 min.
b. Centrifuge at 2000 rpm for 5 minutes, discard the TUNEL reaction solution, add an appropriate amount of 0.1% Triton X-100 (prepared in PBS, containing 5 mg/mL BSA) to gently resuspend the cells, and wash twice for 5 minutes each time, so that the 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.

I. Experimental materials (self supplied)
  PBS buffer (1× , pH~7.4)
0.2% Triton X-100 (prepared in PBS)
0.1% Triton X-100 (prepared in PBS, Containing 5 mg/mL BSA)
4% paraformaldehyde (PBS)
immunohistochemical pen
deparaffinizing solvent (paraffin section samples)
reagents related to paraffin section processing
anti-fluorescence quenching tablet
ddH₂O
II. Experimental design
A. Positive control:
DNase I treatment was used to prepare positive control slides. DNase I can digest single - or double-stranded DNA to produce monodeoxynucleotides or single - or double-stranded oligodeoxynucleotides, which artificially causes apoptosis.
B. Negative control:
TUNEL Reaction Buffer without TdT Enzyme was used and ddH₂O was used instead of TdT Enzyme.
C. Experimental treatment groups.
D. Experimental control group.
III. Experimental procedures :
1. Sample preparation :
(1) for adherent cells or cell smears
      a. Rinse 1 time with PBS.
      Note: If there is concern that the cells of the cell smear are not attached firmly, the sample can be dried to make the cells attach more firmly.
      b. Fixed: add appropriate amount of 4% paraformaldehyde (PBS preparation), 4° C fixed for 30 min. Wash twice with PBS.
      c. Permeabilization: Add appropriate amount of 0.2% Triton X-100 (prepared in PBS) and permeabilize for 20 min at room temperature. Rinse twice with PBS.
      d. Turn step 2. TUNEL reaction .

(2) for suspension cells or cell suspensions
      a. Collect cells (3-5× 10⁶cells), centrifuged at 1000 rpm for 5 min, and washed twice with PBS.
      b. Fixation: Cells were fully resuspended by adding an appropriate amount of 4% paraformaldehyde (prepared in PBS) and fixed at 4 ° C for 30 min. The cells were centrifuged at 2000 rpm for 5 min and washed twice with PBS.
      c. Permeabilization: Add appropriate amount of 0.2% Triton X-100 (prepared in PBS) and permeabilize for 20 min at room temperature. The mixture was centrifuged at 2000 rpm for 5 min and washed twice with PBS.
      d. Turn step 2. TUNEL reaction .

(3) Paraffin tissue sections
      a.  Deparaffinization and hydration: Put the sliced samples in xylene I (10 min) in turn. 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 was rinsed for 5 min and twice.
      Note: Xylene is toxic and volatile, do this in a fume hood.
      b. Blot the liquid around the sliced sample with filter paper, and circle the sample outline with an IHC pen for downstream permeability and labeling.
      Note: If the outline circle of immunohistochemical strokes is found to be destroyed in the subsequent experimental operation, it should be repaired in time.
      c. Permeabilization: at a ratio of 1:100, 2 mg/mL of Proteinase K solution was diluted with PBS to a final concentration of 20 µ g/mL, add 100 &micro droppers to each sample; L, so that the solution covered the entire sample area and incubated for 20 min at 20-37 ° C.
      Note: Proteinase K can penetrate the cell membrane and nuclear membrane, so that the staining reagents in the subsequent steps can fully enter the nucleus for reaction and improve the labeling efficiency. Too long of the incubation time will increase the risk of the tissue section falling off the carrier plate in the subsequent washing step, and too short of the incubation time may cause inadequate permeability treatment and affect the labeling efficiency. In order to obtain better results, the concentration, incubation time and temperature of Proteinase K should be optimized according to different types of tissue samples.
      d. Rinse the sections twice with PBS for 5 min each time, blot off excess liquid with filter paper, and keep the processed sample moist in a wet box.
      Note: This step must wash the Proteinase K clean, otherwise it will seriously interfere with the subsequent labeling reaction.
e. Turn step 2. TUNEL reaction .

(4) Frozen tissue sections
      a. Fix: Remove frozen sections and return to room temperature. Add appropriate amount of 4% paraformaldehyde (prepared in PBS) and fix at room temperature for 30 min. Rinse twice with PBS for 10 min each time.
      Note: If you worry about formaldehyde cleaning is not clean, affect the final dyeing effect. After formaldehyde fixation is completed, an appropriate amount of 2 mg/mL glycine can be added to clean for 10 min to neutralize the residual fixative, and then clean with PBS.
      b. Blot the liquid around the sliced sample with filter paper, and circle the sample outline with an IHC pen for downstream permeability and labeling.
      Note: If the outline circle of immunohistochemical strokes is found to be destroyed in the subsequent experimental operation, it should be repaired in time.
      c. Permeabilization: at a ratio of 1:100, 2 mg/mL of Proteinase K solution was diluted with PBS to a final concentration of 20 µ g/mL, add 100 &micro droppers to each sample; L, so that the solution covered the entire sample area and incubated for 20 min at 20-37 ° C.
      Note: Proteinase K can penetrate the cell membrane and nuclear membrane, so that the staining reagents in the subsequent steps can fully enter the nucleus for reaction and improve the labeling efficiency. Too long of the incubation time will increase the risk of the tissue section falling off the carrier plate in the subsequent washing step, and too short of the incubation time may cause inadequate permeability treatment and affect the labeling efficiency. To get a better result, Proteinase K concentration, incubation time and temperature optimization should be carried out according to the different types of tissue samples.
      d. Rinse the sections twice with PBS for 5 min each time, blot off excess liquid with filter paper, and keep the processed sample moist in a wet box.
        Note: this step must be to Proteinase K washing clean, otherwise it will seriously interfere with subsequent tag response.
        e. Turn step 2. TUNEL reaction .

(5) Positive processing (this step is only carried out for positive control, and the other samples are directly carried out for TUNEL reaction step)
        A. 10&times with ddH₂O at a ratio of 1:10; DNase I dilute Buffer into 1 & times; DNase I Buffer for later use.
        B. add 100 & micro; L 1× DNase I Buffer to the processed samples, sample cover all areas, balance 5 min at room temperature.
        C. use 1 & times; DNase I Buffer to 1:100 diluted DNase I (2 U / & mu; L) to finish working liquid concentration 20 U/mL.
        D. to Buffer, to join 100 & mu; L DNase I working solution at a concentration of 20 U/mL was incubated for 10 min at room temperature.
        e. Discard the DNase I working solution and wash twice with PBS.
        f. Turn step 2. TUNEL reaction .
2, TUNEL reaction
(1) Prepare TUNEL reaction solution (ready to use) :