Biotin TUNEL Apoptosis Detection Kit
Biotin TUNEL Apoptosis Detection Kit
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Product Specification
| Usage |
1. Experimental materials (self-prepared) PBS buffer (1 ×, pH ~ 7.4) 0.2% Triton X-100 (formulated in PBS) Reagents related to paraffin section processing 4% Paraformaldehyde (PBS formulated) Immunohistochemical pen 0.3% H2O2(Freshly formulated in PBS) Neutral resin ddH2O 2. Experimental design A. Positive control (optional): 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 (optional): Biotin TUNEL Reaction Buffer without TdT Enzyme was used with 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 (prepared with PBS) and fix at room temperature for 30 minutes. 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. Blocking: add about 100 μL of 0.3% H per well2O2Solution (freshly prepared with PBS), allowed to fully cover the cells, blocked at room temperature and protected from light for 30 min to inactivate the endogenous catalase in the cells, and then washed twice with PBS. e. 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 0.2% 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. Blocking: add about 100 μL of 0.3% H per well2O2Solution (freshly prepared with PBS), resuspended cells were gently aspirated, blocked at room temperature and protected from light for 30 minutes to inactivate endogenous catalase in cells, and then washed twice with PBS. e. Go to step 2. TUNEL reaction. (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. Note: Proteinase K must be cleaned during this step, otherwise it will seriously interfere with the subsequent labeling reaction. e. Blocking: add an appropriate amount of 0.3% H2O2The solution (freshly prepared in PBS) was incubated at room temperature for 30 min to inactivate the endogenous catalase in the sections. F. Rinse the sections twice with PBS for 5 min each time, aspirate the excess liquid with filter paper, and place the processed sample in a wet box to keep it moist. g. Go to step 2. TUNEL reaction. (4) Frozen tissue section A. Fixation: Take out 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. Note: Proteinase K must be cleaned during this step, otherwise it will seriously interfere with the subsequent labeling reaction. e. Blocking: add an appropriate amount of 0.3% H2O2The solution (freshly prepared in PBS) was incubated at room temperature for 30 min to inactivate the endogenous catalase in the sections. F. Rinse the sections twice with PBS for 5 min each time, suck off the excess liquid with filter paper, and place the treated sections in a wet box to keep them moist. g. Go to step 2. TUNEL reaction. (5) Positive treatment (only positive control is subjected to this step, other samples are directly subjected to TUNEL reaction step) A. Using 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. Go to step 2. TUNEL reaction. 2. TUNEL reaction (1) Prepare TUNEL reaction solution (ready for use):
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 it on the sample, which can prevent the evaporation of TUNEL reaction solution and make the TUNEL reaction solution evenly cover the sample. (3) Discard the TUNEL reaction solution and wash it twice with PBS. 3. Preparation of Streptavidin-HRP working solution and DAB color development solution (1) Preparation of Streptavidin-HRP working solution (ready to use):
(2) Preparation of DAB chromogenic solution (ready for use):
(1) Add 50 μL of Streptavidin-HRP working solution to each sample and incubate at 37 ℃ for 30 min. Note: 50μL Streptavidin-HRP working solution is suitable for smear, section or 96-well plate (other different well plates can appropriately adjust the volume of Streptavidin-HRP working solution to cover cells). To prevent evaporation of the Streptavidin-HRP working fluid, it is recommended to coat the samples with an anti-evaporation film. (2) Discard the Streptavidin-HRP working solution and wash it twice with PBS. (3) Add 50μL of DAB color development solution to each sample, incubate at room temperature for 5 minutes or control the staining time under a microscope according to the color development. Note: If the color development is very strong, the color development can be stopped in less than 5 minutes. If the color development is very weak, the color development time can be appropriately extended, or even the color development can be performed overnight. (4) Discard the DAB color solution and wash it twice with PBS. (5) (Optional) Add an appropriate amount of hematoxylin staining solution or methyl green staining solution for nucleus staining. The staining solution was discarded and washed twice with PBS. (6) (Optional) Slice sealing: Immerse the slices in pure water, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol and absolute ethanol for 5 minutes in turn, and finally put the slice samples in a dyeing tank. Soak in fresh xylene, and transparent treatment twice for 5 minutes each time. After the dehydration was complete, the liquid around the sections was wiped away, 50 μL of neutral resin was added dropwise to each sample, a coverslip was covered, and the coverslip was gently tapped with the blunt end of the tweezers to remove air bubbles to complete the sealing. (7) Use filter paper to suck off excess liquid, add 100μL PBS to the sample area to keep the sample moist, and immediately analyze the sample under an optical microscope. |
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| Experiment Reagent Instrument | PBS,Paraformaldehyde,Triton X-100,Methanol,H2O2,Hematoxylin (optional) |
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| Description |
When cells undergo apoptosis, some DNA endonucleases will be activated. These endonucleases will cut off the genomic DNA between nucleosomes and produce DNA fragments of 180 bp to 200 bp, which shows the specific Ladder Ladder map presented in agarose gel electrophoresis. When genomic DNA double-stranded or single-stranded breaks, a large number of sticky 3 '-OH terminals will occur, which can be combined with Biotin-dUTP under the catalysis of deoxyribonucleotide terminal transferase (TdT), so as to directly detect apoptotic cells through light microscopy. This method is called Terminal-deoxynucleotidyl transfer mediated nick end labeling (TUNEL). Since normal or proliferating cells have little DNA breakage, there is no 3 '-OH formation and little can be stained. Tunel method can stain complete single apoptotic nuclei or apoptotic bodies in situ, accurately reflect the typical biochemical and morphological characteristics of apoptosis, and can detect a very small number of apoptotic cells. Therefore, it is widely used in the research of apoptosis.
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| General Notes | 1. For your safety and health, please wear a lab coat and disposable gloves. 2. Sodium azide has an inhibitory effect on HRP. Do not use reagents containing sodium azide in the experiment. |
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| Storage Temp. | This product should be stored at-20 ℃ protected from light; Avoid repeated freezing and thawing. This product can be stored for up to 12 months under recommended conditions. |
