TUNEL BrightGreen Apoptosis Detection Kit A112

During apoptosis, intracellular specific endonucleases are activated, chromatin DNA is specifically cleaved between nucleosomes, and DNA is degraded into integer multiples of about 180 -200 bp fragments. The 3'-hydroxyl (3'-OH) end generated by DNA fragmentation can bind to fluorescein-12-deoxyuridine triphosphate (FITC-12-dUTP) under the action of terminal deoxynu-cleotidyl transferase (TdT). FITC-12-dUTP-labeled DNA can be directly observed by fluorescence microscopy or quantitatively analyzed by flow cytometry to reflect the level of apoptosis. The BrightGreen Labeling Mix contains FITC-12-dUTP...

07/Mechanism & Workflow Frozen Sections Adherent Cells Suspension Cells Sample Preprocessing Paraffin Sections Wash the samples 2 - 3 times Equilibrate 1 × Equilibration Buffer: 100 μl (room temperature, 10 - 30 min) ddH2O 34 μl 5 × Equilibration Buffer 10 μl BrightGreen Labeling Mix 5 μl Recombinant TdT Enzyme 1 μl Stain (counterstaining at room temperature for 5 min) Wash the samples 2 - 3 times Analyze sample

08/Tissue Section/Adherent Cell Experiment Process Please read this instruction carefully before the experiment. This protocol is suitable for the apoptosis detection of paraffin-embedded tissue sections, frozen tissue sections, and adherent cells. Each sample should be carried out in strict accordance with its operating procedures. For suspension cell detection, please refer to 09/Suspension Cell Experiment Process. 08-1/Sample Preprocessing ◇Paraffin-embedded tissue sections 1. Immerse the paraffin tissue sections in xylene at room temperature to completely remove the paraffin (two times for...

3. Dilute DNase I (2 U/jl) with 1 x DNase I Buffer to a final concentration of 20 U/ml. 4. Gently aspirate off excess liquid, add 100 |jl of 20 U/ml DNase I solution, and incubate at room temperature for 10 min. 5. Gently aspirate off the excess liquid, then thoroughly immerse and rinse the covers-lips/slides in a staining jar with 1 x PBS for 2 - 3 times. ▲ Positive control slides must use a separate staining jar. Residual DNase I on positive control slides may cause false positive signals in experimental groups. 1. Dilute 5 x Equilibration Buffer to 1 x Equilibration Buffer with ddhhO at a...

9. Wash the samples, immerse the slides in 1 × PBS solution at room temperature (three times for 5 min each). 10. Gently aspirate off excess liquid and add 100 μl of 20% glycerol in 1 × PBS to the sample area to keep the sample moist. 11. Analyze samples immediately under a fluorescence microscope using a standard fluorescence filter to observe green fluorescence at 520 ± 20 nm; red fluorescence at >620 nm for PI, or blue fluorescence at 460 nm for DAPI. ▲If necessary, the samples can be mounted and stored overnight at 4℃ in the dark. PI/DAPI stains both apoptotic and non-apoptotic cells red/blue....

08-5/Positive Treatment (only the positive control is performed this step, and other samples are directly subjected to 08-6/Labeling and Detection) After permeabilization, samples can be treated with DNase I to prepare positive controls. ▲ DNase I treats fixed cells will cause chromosomal DNA breaks, resulting in many labelable DNA 3' ends. The following processes generally results in green fluorescence in most cells treated. 1. Dilute 10 x DNase I Buffer with dd-hO at a ratio of 1:10 to 1 * DNase I Buffer for use. ▲ Prepare 200 pl of 1 * DNase I Buffer for each sample. 2. Add 100 pl 1 * DNase...

6. Gently remove excess liquid, wash twice with fresh 1 × PBS solution, and incubate at room temperature for 5 min each time. ▲To reduce the background, after washing the sample with 1 × PBS, it can be washed again with PBS containing 0.1% Triton X-100 and 5 mg/ml BSA (three times for 5 min each), which can remove the free unreacted label. 7. Gently wipe off the 1 × PBS solution around and behind the sample with absorbent paper. 8. Counterstain the sample in the dark with a freshly prepared 1 μg/ml PI solution in 1 × PBS or a 2 μg/ml DAPI solution freshly prepared in 1 × PBS. Place at room temperature...

3. Gently remove excess fluid and carefully blot the fluid around the specimen on the coverslips/smears with absorbent paper. ▲ The outline of the sample distribution can be drawn around the sample with a paraffin crayon or a hydrophobic pen, which is convenient for downstream permeabilization, equilibration and labeling. ▲ During the experiment, do not let the sample dry. Keep the treated samples moist in a wet box. 4. Dilute 2 mg/ml Proteinase K solution 1:100 in 1 x PBS to a final concentration of 20 pg/ml. Drop 100 pl of diluted Proteinase K solution on each sample to cover the entire sample...

2. Add 100 |jl 1 x Equilibration Buffer dropwise to make it completely cover the sample area to be tested, and equilibrate at room temperature for 10 - 30 min. 3. Prepare the TdT reaction mixture according to the table below in the dark during equilibration. Components Negative Control Positive Control/Sample ▲ For sma|| ce|| covers|ips/s|ides, the required vo|ume is 50 p|, mu|tip|y 50 p| by the number of experimenta| and positive control reactions to determine the total volume of TdT reaction mixture required. For samples with larger surface areas, the reagent volume can be proportionally increased....

09/Suspension Cell Experiment Process 1. Wash 3 x 106 to 5 x 106 cells with PBS twice, centrifuge at 4°C 1,800 rpm (300 x g) for 5 min each time. Then resuspend with 0.5 ml 1 x PBS. 2. Add 5 ml paraformaldehyde solution (concentration of 1%) prepared in 1 x PBS, and incubate at 4C for 20 min for cell fixation. 3. Centrifuge at 4C 1,800 rpm (300 x g) for 5 min, discard the supernatant, and resuspend the cells with 5 ml of 1 x PBS. 4. Repeat centrifugation and resuspend cells with 0.5 ml 1 x PBS. 5. Add 5 ml Triton X-100 solution (concentration of 0.2%) prepared in 1 x PBS, and permeabilize for...