TUNEL Apoptosis Assay Kit (HRP-DAB)

SKU:

AKES076-20

Product Type:

Assay

Detection Method:

Colorimetric

Research Area:

Apoptosis

TUNEL 1-Step Kits

Description

Description

Background

Assay Genie TUNEL Apoptosis Assay Kit is a highly sensitive, rapid and simple method for cell apoptosis detection. After Biotin-dUTP labeling and DAB staining, cell apoptosis can be detected under ordinary optical microscopy.

This kit is developed to detect tissue (Paraffin embedding, freezing and ultrathin section) and cells (Cell smears, cell climbing smears) in situ apoptotic detection.

Detection Principle

When cells undergo apoptosis, endonuclease enzymes are activated to cleave the genomic DNA between nucleosomes. When genomic DNA is cleaved, the exposed 3'-OH can be added with a biotinylated dUTP (Biotin-dUTP) due to the catalysis of Terminal Deoxynucleotidyl Transferase (TdT), followed by the binding of horseradish Peroxidase-labeled Streptavidin (Streptavidin-HRP). Thereafter, through the HRP catalysis, the apoptotic cells are marked by DAB, which can be detected by ordinary microscopy. This is the principle of TUNEL (TdT-mediated dUTP Nick-End Labeling) for detecting apoptosis.

Kit Components:

Components	20 T	50 T	100 T	Storag
TdT Equilibration Buffer	1.0 mL	1.25 mLx2	5.0 mL	-20°C
TdT Enzyme	80 μL	200 μL	400 μL	-20°C
Proteinase K (50 ×)	40 μL	100 μL	200 μL	-20°C
Streptavidin-HRP	10 μL	25 μL	50 μL	-20°C
Biotin-dUTP	20 μL	50 μL	100 μL	-20°C
DAB Concentrate (20 ×)	200 μL	500 μL	1 mL	-20°C
DAB Substrate	4 mL	10 mL	2× 10 mL	-20°C

Reagents needed but not included:

1. Cell Sample
- Fixative Buffer (Polyformaldehyde dissolved in PBS with final concentration of 4%)
- Blocking Buffer (H2O2 dissolved in Deionized water with final concentration of 3%)
- Permeablilization Buffer (Triton-100 dissolved in 0.1% citrate sodium with final concentration of 0.1%)

2. Paraffin Embedding
- Xylene, ethanol, PBS
- Blocking Buffer (H2O2 dissolved in Deionized water with final concentration of 3%)

3. Freezing Section
- Fixative Buffer (Polyformaldehyde dissolved in PBS with final concentration of 4%)
- Blocking Buffer (H2O2 dissolved in Deionized water with final concentration of 3%)
- Permeablilization Buffer (Triton-100 dissolved in 0.1% citrate sodium with final concentration of 0.1%).

4. Positive Control
- DNase I

5. Other Reagents
- PBS
- Hematoxylin (nuclear staining buffer)
- Neutral Balsam.

Storage:

Streptavidin-HRP should be stored at 2~8°C, other reagents can be stored at -20°C.

Streptavidin-HRP and 20×DAB Concentrate should be stored in dark.

Cautions:

1. For maximal assay performance, this reagent should be used within 12 months. Avoid freeze / thaw cycles.
2. After washing the slides with PBS, please wipe dry the liquid around the tissue on the slides with absorbent paper.
3. Keep the sample moist during the experiment to prevent the failure of the experiment caused by dry slices.
4. Prepare the TdT Enzyme working solution before use, it can be store on ice for short time, enzyme activity may lose after long-term preservation.
5. If the 20×DAB Concentrate became dark purple, please do not use it.
6. This kit is for research use only. For your safety and health, please wear lab clothes and gloves. Instructions should be followed strictly, changes of operation may result in unreliable results.