Biotin Apoptosis Detection Kit (TUNEL)
Based on 1 Customer Validation
MCE Biotin Apoptosis Detection Kit (TUNEL) can be used to detect apoptosis in cultured cells, as well as in frozen or paraffin sections.
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Storage :
-20°C, 2 years
Keep away from light.
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Description & Advantages
Apoptosis is a form of programmed cell death. Based on the cellular changes during programmed cell death, apoptosis is often classified into early, middle, and later stages. The later stages of apoptosis are characterized by changes in nuclear morphology, chromatin condensation, nuclear envelope degradation, and DNA fragmentation.
The principle of TUNEL (TdT-mediated dUTP Nick-End Labeling) in detecting cell apoptosis is that during apoptosis, certain DNA endonucleases are activated, which cleave genomic DNA between nucleosomes. In normal or proliferating cells, there is almost no DNA breakage, thus 3'-OH formation is scarce, leading to minimal labeling. The exposed 3'-OH can be catalyzed by terminal deoxynucleotidyl transferase (TdT) to incorporate biotin-labeled dUTP, which can conjugate with horseradish peroxidase (HRP) -labeled streptavidin-HRP. Through DAB staining catalyzed by HRP, apoptotic cells can be detected, enabling cell apoptosis detection.
MCE Biotin Apoptosis Detection Kit (TUNEL) can be used to detect apoptosis in cultured cells, as well as in frozen or paraffin sections. It selectively detects apoptotic cells and distinguishes between apoptotic and necrotic cells. Due to the distinct patterns of DNA fragmentation, necrotic cells or non-apoptotic cells with broken DNA strands caused by irradiation and drug therapy will not be identified as apoptotic cells.
Protocol
1. Sample fixation and permeabilization
1. Fix cultured cells with 4% paraformaldehyde for approximately 30 min. For frozen or paraffin sections, complete routine fixation/deparaffinization before proceeding.
2. Wash samples with PBS.
3. Permeabilize the sample with an appropriate permeabilization solution to allow the TdT reaction mixture to access DNA.
2. TUNEL labeling
1. Prepare the TUNEL reaction mixture containing TdT Enzyme and Biotin-dUTP Labeling Mix according to the kit instructions immediately before use.
2. Remove excess PBS and add sufficient TUNEL reaction mixture to cover the sample area.
3. Incubate at 37°C for 60 min.
4. Wash three times with PBS.
3. Detection of the biotin signal
Use an appropriate Streptavidin-based detection system for chromogenic or fluorescent detection, and complete incubation, washing, and imaging according to the selected detection reagent.
4. Controls
Omit TdT Enzyme in the negative control. A positive control may be generated by DNase I treatment before the TUNEL reaction. TUNEL-positive signal indicates apoptotic cells containing DNA strand breaks.
Storage
-20°C, 2 years
Keep away from light.
Attention
1. Please centrifuge the regent to the bottom of the tube before use.
2. As Sodium azide (NaN3) inhibits HRP, the reaction system must not contain NaN3.
3. This product is for R&D use only, not for drug, house hold, or other uses.
4. For your safety and health, please wear a lab coat and disposable gloves to operate.
Components
| Components | HY-K1091A-20 T | HY-K1091A-50 T |
|---|---|---|
| Biotin TUNEL Reaction Buffer | 1 mL | 2 × 1.25 mL |
| TdT Enzyme | 40 μL | 100 μL |
| Streptavidin-HRP | 20 μL | 50 μL |
| Streptavidin-HRP Diluent Buffer | 1 mL | 2 × 1.25 mL |
| DAB Concentrate (20×) | 50 μL | 125 μL |
| DAB Diluent Buffer | 1 mL | 2 × 1.25 mL |
| Coloration Enhancement Buffer (10×) | 100 μL | 250 μL |
| Proteinase K (2 mg/mL) | 40 μL | 100 μL |
| DNase I (2 U/μL) | 5 μL | 13 μL |
| DNase I Buffer (10×) | 100 μL | 260 μL |
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