TUNEL Assay Kit (HRP-DAB)[331]

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      Catalog number:E-CK-A331

      Size:
      • 20T
      • 50T
      • 100T
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      Price: $300

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      Introduction

      Elabscience® 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.


      Components


      Cat.

      Products

      20 Tests

      50 Tests

      100 Tests

      Storage

      E-CK-A331A

      TdT Equilibration Buffer

      1.0 mL

      1.25 mLx2

      5.0 mL

      -20°C

      E-CK-A331B

      TdT Enzyme

      80 μL

      200 μL

      400 μL

      -20°C

      E-CK-A331C

      Proteinase K (50 ×)

      40 μL

      100 μL

      200 μL

      -20°C

      E-CK-A331D

      Streptavidin-HRP

      10 μL

      25 μL

      50 μL

      2~8°C

      E-CK-A331E

      Biotin-dUTP

      20 μL

      50 μL

      100 μL

      -20°C

      E-CK-A331F

      DAB Concentrate (20 ×)

      200 μL

      500 μL

      1 mL

      -20°C

      E-CK-A331G

      DAB Substrate

      4 mL

      10 mL

      2× 10 mL

      -20°C

      Manual

       One Copy



      Reagent Not Included

           1.Cell sample

      Fixative Buffer (Polyformaldehyde dissolved in PBS with final concentration of 4%)(It is recommended to use Elabscience® E-IR-R113).

      Blocking Buffer (H2O2 dissolved in Deionized water with final concentration of 3%)(It is recommended to use Elabscience® E-IR-R115).

      Permeablilization Buffer (Triton-100 dissolved in 0.1% citrate sodium with final concentration of 0.1%)(It is recommended to use Elabscience® E-IR-R122).


      2.Paraffin embedding

      Xylene, ethanol, PBS.

      Blocking Buffer (H2O2 dissolved in Deionized water with final concentration of 3%)(It is recommended to use Elabscience® E-IR-R115).


            3.Freezing section

      Fixative Buffer (Polyformaldehyde dissolved in PBS with final concentration of 4%)(It is recommended to use Elabscience® E-IR-R113).

      Blocking Buffer (H2O2 dissolved in Deionized water with final concentration of 3%)(It is recommended to use Elabscience® E-IR-R115).

      Permeablilization Buffer (Triton-100 dissolved in 0.1% citrate sodium with final concentration of 0.1%)(It is recommended to use Elabscience® E-IR-R122).


            4.Positive control  

                  DNase I

            5.Other reagents  

                PBS(It is recommended to use Elabscience® E-BC-R187).

                Hematoxylin (nuclear staining buffer, It is recommended to use Elabscience® E-IR-R120).

                Neutral Balsam(It is recommended to use Elabscience® E-IR-R118).


      Instructions

      1.  Proteinase K (50 ×)[E-CK-A331C] is concentrated, diluted with PBS to 1 × working solution before use.

      For example: take 10 μL Proteinase K (50 ×)[E-CK-A331C], add to 490 μL PBS, the mixture is 1×Proteinase K working solution.

       

      2.  DAB Concentrate (20 ×)[ E-CK-A331F] is concentrated, diluted with DAB Substrate[ E-CK-A331G] to 1 × working solution before use.

      For example: take 10 μL DAB Concentrate (20 ×)[ E-CK-A331F], add to 190 μL DAB Substrate[ E-CK-A331G], the mixture is 1×DAB working solution.


      Storage

      Streptavidin-HRP [E-CK-A331D] stored at 2~8°C, other reagents stored at -20°C. Streptavidin-HRP[E-CK-A331D] and 20×DAB Concentrate [E-CK-A331F] 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.

      Citations

      1. In Vitro and In Vivo Pretreatment with Selenium Mitigates Tetrahydrocannabinol-Induced Testicular Cell Apoptosis: the Role of AKT and p53 Pathways
        IF: 2.639
        Journal: Biological Trace Element Research(2020)
        DOI: 10.1007/s12011-020-02322-5
        PMID: 32815089
        Products: E-CK-A331 
        Sample type: Cell sample
        Reactivity: Rat
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