Biotin TUNEL apoptosis Kit

Cat. No.: B598358

Overview

When cell apoptosis occurs, some DNA endonucleases will be activated. These endonucleases will cut off genomic DNA between nucleosomes and generate 180 bp to 200 bp DNA fragments, which is shown as a specific ladder map in agarose gel electrophoresis. When genomic DNA undergoes double or single strand breaks, a large number of sticky 3 '- OH terminals are produced, which can bind to biotin dUTP under the catalytic action of deoxyribonucleotide terminal transferase (TdT), and directly detect apoptotic cells through optical microscopy. This method is called terminal deoxyribonucleotide terminal transferase mediated nick end labeling (TUNEL). Due to the almost absence of DNA breakage in normal or proliferating cells, there is no formation of 3 '- OH and it is rarely stained. The Tunel method can perform in situ staining on intact individual apoptotic nuclei or bodies, accurately reflecting the typical biochemical and morphological characteristics of cell apoptosis, and can detect a very small number of apoptotic cells. Therefore, it is widely used in the study of cell apoptosis. This reagent 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.

Component： 

Instruction： 

Experimental materials (self provided)
PBS buffer (1 x, pH~7.4)
0.2% Triton X -100 (PBS formulation)
Related reagents for paraffin section processing
4% paraformaldehyde (prepared with PBS)
Immunohistochemical pen
0.3% H2O2 (PBS freshly prepared)
Neutral resin
. ddH2O
experimental design
A. Positive control (optional): Prepare positive control slides using DNaseI treatment. DNaseI can digest single or double stranded DNA to produce single deoxyribonucleotides or single or double stranded oligodeoxyribonucleotides as endonucleases, artificially causing cell apoptosis.
B. Negative control (optional): Use Biotin TUNEL Reaction Buffer without TdT Enzyme and replace TdTEnzyme with ddH2O.
C. Experimental processing group.
D. Experimental control group.
Experimental steps
1. Sample preparation:
(1) For adherent cells or cell smears
a. Clean once with PBS.
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 room temperature for 30 minutes. Clean twice with PBS.
c. Translucency: Add an appropriate amount of 0.2% Triton X -100 (prepared with PBS) and let it penetrate at room temperature for 20 minutes. Clean twice with PBS.
d. Closed: Add 100 to each hole μ Approximately 0.3% H2O2 solution (freshly prepared with PBS) was prepared and allowed to fully cover the cells. The cells were sealed at room temperature in dark for 30 minutes to inactivate endogenous catalase. Subsequently, the cells were washed twice with PBS.
e. Step 2: TUNEL reaction.
(2) For suspended cells or cell suspensions
a. Collect cells (3-5 x 106 cells), centrifuge at 1000 rpm 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 clean twice with PBS.
c. Translucency: Add an appropriate amount of 0.2% Triton X -100 (prepared with PBS) and let it penetrate at room temperature for 20 minutes. Centrifuge at 2000 rpm for 5 minutes and clean twice with PBS.
d. Closed: Add 100 to each hole μ A 0.3% H2O2 solution (freshly prepared with PBS) of about L was gently blown and aspirated to resuspend the cells. The cells were closed at room temperature in the dark for 30 minutes to inactivate endogenous catalase, and then washed twice with PBS.
e. Step 2: TUNEL reaction.
(3) Paraffin tissue sectioning
a. Dewaxing and hydration: Place the sliced samples sequentially in 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, rinse twice.
Note: Xylene is toxic and volatile. Please perform this operation in a fume hood.
b. Use filter paper to dry the liquid around the sliced sample, and circle the sample contour with an immunohistochemical pen for downstream transparency and labeling.
Note: If it is found that the contour circle of immunohistochemistry strokes is damaged in subsequent experimental operations, it needs to be redrawn in a timely manner.
c. Transparency: Dilute 2 mg/mL of Protein K solution with PBS in a ratio of 1:100 to a final concentration of 20 µ g/mL. Add 100 µ L dropwise to each sample to cover all sample areas. Incubate at 20-37 ℃ for 20 minutes.
Note: ProteinaseK can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to fully enter the nucleus for reaction and improve labeling efficiency. An excessively long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while a too short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To obtain better results, the concentration, incubation time, and temperature of Protein K need to be optimized according to different types of tissue samples.
d. Wash the slices with PBS twice for 5 minutes each time.
Note: Protein K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.
e. Sealing: Add an appropriate amount of 0.3% H2O2 solution (PBS freshly prepared) and incubate at room temperature for 30 minutes to inactivate endogenous catalase in the slices.
f. Wash the slices with PBS twice, 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.
g. Step 2: TUNEL reaction.
(4) Frozen tissue sections
a. Fixation: Take out frozen sections and warm them back to room temperature. Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and fix at room temperature for 30 minutes. Rinse twice with PBS for 10 minutes each time. Note: If you are concerned that formaldehyde cleaning may not be clean enough, it may affect the final dyeing effect. After formaldehyde fixation is completed, an appropriate amount of 2 mg/mL glycine can be added and washed for 10 minutes to neutralize the residual fixing solution, and then PBS cleaning can be carried out.
b. Use filter paper to dry the liquid around the sliced sample, and circle the sample contour with an immunohistochemical pen for downstream transparency and labeling.
Note: If it is found that the contour circle of immunohistochemistry strokes is damaged in subsequent experimental operations, it needs to be redrawn in a timely manner.
c. Transparency: Dilute 2 mg/mL of Protein K solution with PBS in a ratio of 1:100 to a final concentration of 20 µ g/mL. Add 100 µ L dropwise to each sample to cover all sample areas. Incubate at 20-37 ℃ for 20 minutes.
Note: ProteinaseK can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to fully enter the nucleus for reaction and improve labeling efficiency. An excessively long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while a too short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To obtain better results, the concentration, incubation time, and temperature of Protein K need to be optimized according to different types of tissue samples.
d. Wash the slices with PBS twice for 5 minutes each time.
Note: Protein K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.
e. Sealing: Add an appropriate amount of 0.3% H2O2 solution (PBS freshly prepared) and incubate at room temperature for 30 minutes to inactivate endogenous catalase in the slices.
f. Rinse the slices with PBS twice, each time for 5 minutes. Use filter paper to remove excess liquid, and place the processed slices in a wet box to keep them moist.
g. Step 2: TUNEL reaction.
(5) Positive treatment (only the positive control is subjected to this step, and other samples are directly subjected to the TUNEL reaction step)
a. Dilute 10 x DNase I Buffer with ddH2O in a ratio of 1:10 to 1 x DNase I Buffer for later use.
b. Drip 100 µ L of 1xDNase I Buffer onto the processed sample, covering all sample areas, and equilibrate at room temperature for 5 minutes.
c. Dilute DNase I (2 U) with 1 x DNase I Buffer at a ratio of 1:100/ μ L) A working solution with a final concentration of 20 U/mL.
d. Discard the buffer and add 100 μ Incubate DNase I working solution with a concentration of 20 U/mL at room temperature for 10 minutes.
e. Discard DNase I working solution and clean twice with PBS.
f. Step 2: TUNEL reaction.
2. TUNEL reaction
(1) Prepare TUNEL reaction solution (ready to use):

TUNEL Preparation of reaction solution	1 sample	5 sample	10 sample
TdT enzyme	1 μL	5 μL	10 μL
Biotin TUNEL Reaction Buffer	49 μL	245 μL	490 μL
Total volume	50 μL	250 μL	500 μL

(2) Add 50 to each sample μ LTUNEL reaction solution is used to evenly cover the sample with the reaction solution. Incubate at 37 ℃ for 60 minutes.
Note: 50 μ L TUNEL reaction solution is suitable for smear, slicing, or 96 well plates (other different well plates can adjust the volume of TUNEL reaction solution appropriately to cover cells). If the sample to be tested is a smear, slice, or in a 24 well plate, 12 well plate, or 6 well plate, anti evaporation film can be used, or self sealing bags or other appropriate materials can be used to cut circular plastic sheets slightly smaller than the holes, and TUNEL reaction solution can be added dropwise to cover the sample, which can prevent the evaporation of TUNEL reaction solution and make the TUNEL reaction solution evenly cover the sample.
(3) Discard TUNEL reaction solution and clean twice with PBS.
3. Preparation of Streptavidin HRP working solution and DAB colorimetric solution
(1) Preparation of Streptavidin HRP working solution (ready to use):

Streptavidin-HRP Preparation of working fluid	1 sample	5 sample	10 sample
Streptavidin-HRP	1 μL	5 μL	10 μL
Streptavidin-HRP Dilution solution	49 μL	245 μL	490 μL
Total volume	50 μL	250 μL	500 μL

(2) Preparation of DAB color solution (ready to use):

DAB Preparation of color solution	1 sample	5 sample	10 sample
DAB  Concentrate（20×)	2.5 μL	12.5 μL	25 μL
DAB Dilution solution	47.5 μL	237.5 μL	475 μL
Total volume	50 μL	250 μL	500 μL

Attention: (1) Solution A/B must be completely melted and mixed before use.
(2) Do not use buffer solutions containing sodium azide, which is an HRP inhibitor.
(3) The prepared working solution can be stably stored in the dark at 4 ℃ for 5 days, and any precipitation in the working solution does not affect the dyeing process. For working fluids with precipitation, it is recommended to use the supernatant after high-speed centrifugation.
(3) Preparation of DAB color enhancement solution (ready to use):

DAB Preparation of color enhancement solution	1 个样本	5 个样本	10 个样本
Color enhancer（10×)	5 μL	25 μL	50 μL
Distilled water	45 μL	225 μL	450 μL
Total volume	50 μL	250 μL	500 μL

Attention: Solution C must be completely mixed before use.
4. Sample color rendering
(1) Add 50 to each sample μ L Streptavidin HRP working solution, incubated at 37 ℃ for 30 minutes.
Note: 50 μ L Streptavidin HRP working solution is suitable for smear, slicing, or 96 well plates (for other different well plates, the volume of Streptavidin HRP working solution can be adjusted appropriately to cover the cells). To prevent the evaporation of Streptavidin HRP working solution, it is recommended to cover the sample with an anti evaporation film.
(2) Discard Streptavidin HRP working solution and clean twice with PBS.
(3) Add 50 to each sample μ L DAB color solution, incubate at room temperature in dark for 1-30 minutes or longer, depending on the color development of the sample. If there is no background present, continue to incubate until the color reaches the expected depth.
(4) Remove DAB staining solution and rinse the sample 3-5 times with distilled water to stop the color reaction.
(5) (Optional) When the DAB color is lighter or further signal enhancement is required, the color enhancement step can be selected.
(6) (Optional) After the last wash, remove distilled water and add 50 μ L color enhancement solution ensures sufficient coverage of the sample. Incubate at room temperature in dark for 1-30 minutes or longer, depending on the color development of the sample, until the light brown precipitate generated by DAB at the epitope turns into a dark brown precipitate without significant non-specific or background coloring.
(7) (Optional) Remove the color enhancement solution and rinse the sample 3-5 times with distilled water to terminate the enhanced color reaction.
(8) (Optional) Add an appropriate amount of hematoxylin staining solution or methyl green staining solution for cell nucleus staining. Discard the staining solution and wash twice with PBS.
(9) (Optional) Slice sealing: Add 50 drops to each sample μ L neutral resin, cover with a cover glass, gently tap the cover glass with the blunt end of tweezers to remove bubbles and ensure complete sealing.
(10) Use filter paper to remove excess liquid and add 100 to the sample area μ Keep the sample moist with PBS and immediately analyze the sample under an optical microscope.

Matters needing attention:

1.Components A, E, F, and G need to avoid light and avoid repeated freezing and thawing ; 

2.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments ; 

3.Sodium azide has an inhibitory effect on HRP, do not use reagents containing sodium azide in the experiment ; 

4.Please wear masks and gloves when using components A, E, F and G.If you touch the skin, please rinse immediately with a large amount of water ; 

5.For your safety and health, please wear experimental clothes and disposable gloves.

Scope of application:

Late apoptosis detection, TUNEL Kit

Storage and Shipping

Storage

Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads