Item Number：B1013/4/5/6

Storage conditionsStore at -20 ℃, component A should be kept away from light and avoid repeated freezing and thawing.

Product components：

Product Introduction

A remarkable feature of cell apoptosis is the degradation of cell chromosome DNA, which is very specific and regular. The resulting DNA fragments of different lengths are integral multiples of 180 bp-200 bp, which is shown as a specific ladder pattern in agarose gel electrophoresis. This kit uses TUNEL method and Terminal Deoxyribonucleoside Transferase (TdT) to catalyze the incorporation of YF into the 3 ′ - OH end of fragmented DNA in apoptotic cells ®/ Cy-dUTP，YF ®/ Cy dUTP labeled DNA can be directly observed under a fluorescence microscope or quantified using a flow cytometer. The TUNEL method can selectively detect apoptotic cells, rather than necrotic cells or cells with DNA strand breaks caused by irradiation and drug therapy. The labeled dUTP (such as digoxin dUTP, biotin dUTP) can be directly detected in situ, which is a faster and more direct detection method.

This kit has a wide range of applications and can be used to detect cell apoptosis in frozen or paraffin sections, as well as in cultured adherent or suspended cells. Selective detection of apoptotic cells, rather than necrotic cells or cells with DNA strand breaks caused by irradiation and drug therapy. This reagent kit detects cell apoptosis in a short time, requiring only one staining reaction and washing for detection.

Usage

Experimental materials (self provided)

PBS buffer (pH~7.4)

0.4% Triton X-100 (PBS preparation)

0.1% Triton X-100 (prepared in PBS, containing 5 mg/mL BSA)

4% paraformaldehyde (prepared with PBS)

Immunohistochemistry pen

Dewaxing solvent (paraffin section sample)

Paraffin section processing related reagents

Anti fluorescence quenching sealing agent

ddH2O

Experimental design

A. Positive control

Prepare positive control slides using DNaseI treatment. DNase I can digest single or double stranded DNA exposed to the 3 '- OH end, artificially causing cell apoptosis. Just do one experiment each time. (Used to verify if there are any issues with the operation and reagent kit in this experiment)

B. Negative control

Use without TdT Enzyme TUNEL Reaction Buffer， Replace TdT with ddH2O Enzyme。 (Mainly to exclude non-specific staining caused by cell apoptosis, manipulation process, etc.; and adjust the exposure intensity for photography.)

C. Experimental processing group

The experimental group operated normally according to the instructions.

D. Experimental control group

The experimental group operated normally according to the instructions.

Experimental Procedure

1. Sample preparation:

(1) For adherent cells or cell smears

a. Wash with PBS once.

Note: If you are concerned that the cells on the cell smear may not adhere firmly, you can dry the sample to make the cells adhere more firmly.

b. Fixed: Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and fix at 4 ℃ for 30 minutes. Wash twice with PBS.

c. Permeation: Add an appropriate amount of 0.4% TritonX-100 (prepared in PBS) and let it permeate at room temperature for 20 minutes. Wash twice with PBS.

d. Step 2: TUNEL reaction.

(2) For suspended cells or cell suspensions

a. Collect cells (3-5 × 10)⁶Centrifuge at 1000rpm for 5 minutes and wash twice with PBS.

b. Fixation: Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and resuspend the cells thoroughly. Fix at 4 ℃ for 30 minutes. Centrifuge at 2000 rpm for 5 minutes and wash twice with PBS.

c. Permeation: Add an appropriate amount of 0.4% TritonX-100 (prepared in PBS) and let it permeate at room temperature for 20 minutes. Centrifuge at 2000 rpm for 5 minutes and wash twice with PBS.

d. Step 2: TUNEL reaction.

(3) Paraffin tissue section

a. Place the paraffin sections on the slicing rack and bake them in a 60 ℃ oven for 60 minutes.

b. Dewaxing and hydration: Place the sliced samples in xylene I (10 minutes), xylene II (10 minutes), 100% ethanol I (5 minutes), 100% ethanol II (5 minutes), 95% ethanol (5 minutes), 90% ethanol (5 minutes), 80% ethanol (5 minutes), 70% ethanol (5 minutes), and ddH2O for 5 minutes, rinsing twice.

Note: Xylene is toxic and volatile. Please perform this operation in a fume hood.

c. Use filter paper to absorb the surrounding liquid of the sliced sample, and circle the sample outline with an immunohistochemical pen for downstream permeability and labeling.

Note: If the contour circle of immunohistochemistry strokes is found to be damaged during subsequent experimental operations, it should be redrawn in a timely manner.

d. Transparency: Dilute 2mg/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 to cover the entire sample area. Incubate at 37 ℃ for 30 minutes.

Note: Proteinase K can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to fully enter the nucleus for reaction and improve labeling efficiency. Long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To achieve better results, the concentration of Proteinase K, incubation time, and temperature need to be optimized according to different types of tissue samples.

e. Soak the sliced sample in 1 × PBS and rinse three times, each time for 5 minutes. Use filter paper to remove excess liquid, and place the processed sample in a wet box to keep it moist.

Note: Proteinase K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.

f. Step 2: TUNEL reaction.

(4) Frozen tissue sections

a. Fixed: Remove frozen sections and warm them back to room temperature. Immerse the sliced sample in 4% paraformaldehyde (prepared in PBS) and fix at room temperature for 30 minutes. Soak the sliced sample in 1 × PBS and rinse three times, each time for 10 minutes.

Note: If you are concerned that formaldehyde cleaning may not be thorough and affect the final dyeing effect. After formaldehyde fixation is completed, add an appropriate amount of 2mg/mL glycine for 10 minutes to neutralize the residual fixative, and then wash with PBS.

b. Use filter paper to absorb the surrounding liquid of the sliced sample, and circle the sample outline with an immunohistochemical pen for downstream permeability and labeling.

Note: If the contour circle of immunohistochemistry strokes is found to be damaged during subsequent experimental operations, it should be redrawn in a timely manner.

c. Transparency: Dilute 2mg/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 to cover the entire sample area. Incubate at 37 ℃ for 20 minutes.

Note: Proteinase K can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to be usedFully enter the nucleus for reaction and improve labeling efficiency. Long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To achieve better results, the concentration of Proteinase K, incubation time, and temperature 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, the sample can be immersed in 1% Triton X-100 (PBS preparation) and incubated at room temperature for 3-5 minutes; Afterwards, immerse the sliced samples in 1 × PBS and rinse three times for 5 minutes each time.

d. Soak the sliced sample in 1 × PBS and rinse three times, each time for 5 minutes. Use filter paper to remove excess liquid, and place the processed sample in a wet box to keep it moist.

Note: Proteinase K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.

e. Step 2: TUNEL reaction.

(5) Positive treatment (only positive controls undergo this step, other samples undergo TUNEL reaction step directly)

a. Dilute 10 × DNase I Buffer with ddH2O at a ratio of 1:10 to 1 × DNase I Buffer for later use.

b. Add 100 µ L of 1 × DNase I Buffer dropwise onto the processed sample, covering the entire sample area, and equilibrate at room temperature for 5 minutes.

c. Dilute DNase I (2 U/μ L) with 1 x DNase I Buffer at a ratio of 1:100 to a final concentration of 20 U/mL.

d. Discard the buffer and add 100 μ L of DNaseI working solution with a concentration of 20U/mL, and incubate at room temperature for 15 minutes.

e. Discard DNase I working solution and wash twice with PBS.

f. Step 2: TUNEL reaction.

2. TUNEL reaction

(1) Prepare TUNEL reaction solution (ready to use)

(2) For adherent cells, cell smears, or tissue sections

a. Add 50 μ L of TUNEL reaction solution to each sample to evenly cover the sample. The appropriate time for incubation at 37 ℃ in the dark is 15-30 minutes for cell staining and 1 hour for tissue staining.

Note: 50 μ L TUNEL reaction solution is suitable for smears, sections, or 96 well plates (other different well plates can adjust the volume of TUNEL reaction solution appropriately to cover the cells). If the sample to be tested is a smear, slice, or in a 24 well, 12 well, or 6-well plate, an anti evaporation film can be used, or self sealing bags or other suitable materials can be used to cut into circular plastic sheets slightly smaller than the well. After adding TUNEL reaction solution dropwise, cover the sample to prevent TUNEL reaction solution from evaporating and evenly cover the sample.

b. Discard the TUNEL reaction solution, wash twice with PBS, and then use 0.1% more Triton X-100 Wash 3 times with PBS (containing 5mg/mL BSA) for 5 minutes each time, so that free unreacted markers can be removed more thoroughly.

c. (Optional) Add an appropriate amount of DAPI staining solution with a concentration of 5 μ g/mL to each sample, and keep it away from light at room temperatureincubation5min。 After staining, discard the DAPI staining solution and wash twice with PBS for 5 minutes each time.

d. (Optional) Slice sealing: Add 20 μ L of anti fluorescence quenching sealing agent (anti fluorescence) dropwise to each sampleQuenching and sealing agents may not be suitable for certain dyes. It is recommended to conduct pre experimental testing and matching before the experimentCover the slide with a lid and gently tap it with the blunt end of tweezers to remove any air bubbles and ensure the seal is intactcomplete.

e. Use filter paper to remove excess liquid and add 100 μ L to the sample area PBS keeps the sample moist,Immediately observe under a fluorescence microscope.

(3) For suspended cells or cell suspensions

a. Add 50 μ L TUNEL reaction solution to each sample tube and gently resuspend the cells. Incubate at 37 ℃ in the dark15-30min。 Gently resuspend cells every 10 minutes using a micropipette.

b. Centrifuge at 2000 rpm for 5 minutes, discard the TUNEL reaction solution, and use 0.1% Triton X-100(PBS preparation, containing 5mg/mL) Wash twice with BSA, each time for 5 minutes, and swim like thisUnreacted markers can be removed relatively cleanly.

c. Add 100 μ L of DAPI staining solution with a concentration of 5 μ g/mL to each sample tube and incubate at room temperature in the dark for 5 minutes.

d. Add 400 μ L Resuspend cells in PBS and immediately detect them using a flow cytometer or smear themObserve under a fluorescence microscope.

Precautions

Before use, please centrifuge the product instantly to the bottom of the tube before conducting subsequent experiments.

When the staining background is heavy or non-specific staining is obvious, the staining time can be appropriately reduced.

3. It is recommended to add negative and positive control groups during the experiment.

When using component A, please wear a mask and gloves. If it comes into contact with the skin, please rinse immediately with plenty of water.

5. Fluorescent dyes all have quenching problems, please try to avoid light as much as possible to slow down fluorescence quenching.

6. This product is for scientific research only and should not be used for clinical diagnosis or treatment, food or medicine, or stored in ordinary residential areas.

For your safety and health, please wear lab clothes and disposable gloves when operating.