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    tunel-assay-kit-hrp-dab-ab206386.pdf

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TUNEL Assay Kit - HRP-DAB (ab206386)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (2) Submit a question References (83)

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TUNEL assay staining
  • ab206386
  • ab206386

Key features and details

  • Assay type: Cell-based (qualitative)
  • Detection method: Colorimetric
  • Assay time: 5 hr
  • Sample type: Adherent cells, Tissue

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Overview

  • Product name

    TUNEL Assay Kit - HRP-DAB
    See all DNA fragmentation kits
  • Detection method

    Colorimetric
  • Sample type

    Tissue, Adherent cells
  • Assay type

    Cell-based (qualitative)
  • Assay time

    5h 0m
  • Product overview

    TUNEL Assay Kit - HRP-DAB ab206386 allows the recognition of apoptotic nuclei in paraffin-embedded tissue sections, frozen tissue sections, or in preparations of single cell suspensions fixed on slides.


    The TUNEL assay is used to detect DNA fragmentation, such as in apoptosis. It uses terminal deoxynucleotidyl transferase (TdT) to catalyze the incorporation of deoxynucleotides at the free 3'-hydroxyl ends of fragmented DNA. The deoxynucleotides are then labeled in a variety of ways for detection of the degree of DNA fragmentation.


    In this TUNEL assay protocol:
    - terminal deoxynucleotidyl Transferase (TdT) binds to exposed 3’-OH ends of DNA fragments generated in response to apoptotic signals and catalyzes the addition of biotin-labeled deoxynucleotides
    - biotinylated nucleotides are bound with a streptavidin-horseradish peroxidase (HRP) conjugate
    - diaminobenzidine (DAB) reacts with the HRP labeled sample to generate an insoluble colored (brown) substrate at the site of DNA fragmentation 
    - counterstaining with methyl green aids in the evaluation of normal and apoptotic cells


    ==

  • Notes

    This kit is designed for chromogenic TUNEL staining with HRP and DAB. It was previously called In situ Apoptosis Detection Kit (DAB).

    To use FITC (Ex/Em = 495/519 nm) as a label, we recommend TUNEL Assay Kit - FITC (ab66108).

    To use BrdU-Red (Ex/Em = 488/576nm) as a label, we recommend TUNEL Assay Kit - BrdU-Red (ab66110).

    Find out more about the TUNEL method in the TUNEL staining / TUNEL assay guide.

    The methyl green counterstain is water soluble and so a non-aqueous/organic mounting media needs to be used.

    How other researchers have used HRP-DAB TUNEL Assay Kit ab206386

    This TUNEL assay kit has been used in publications in a variety of sample types, including:
    - Human: LX-2 cell cultures1 
    - Mouse (paraffin-embedded sections): abdominal aortic aneurysm lesion2, thyroid gland3, lung tissue4, heart5, liver6, ovary tissue7, skin8, liver9
    - Rat: paraffin embedded heart10, brain tissue11
    - Other: paraffin embedded human xenograft tumors in mice12, paraffin-embedded tissue from nude mice injected with human pancreatic cancer cell line13

    References: 1-Park SM et al 2017, 2-Li et al 2019, 3-Iglesias-Osma MC et al 2019, 4-Yuan X et al 2018, 5-Ravi V et al 2018, 6-Jadhav K et al 2018, 7-Gao Y et al 2017, 8-Shin JM et al 2017, 9-Kim MH et al 2017 and 2016, 10-Zhang J et al 2018, 11-Xiao B et al 2017, 12-Nielsen CF et al 2017, 13-Dauer et al 2019

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 30 slides 60 slides
    25X Conjugate 1 x 150µl 1 x 300µl
    Blocking Buffer 1 x 12ml 1 x 24ml
    DAB Solution 1 (DAB Concentrate) 1 x 150µl 1 x 300µl
    DAB Solution 2 (Substrate Reaction Buffer) 1 x 4ml 1 x 8ml
    Methyl Green Counterstain 1 x 3.5ml 2 x 3.5ml
    Proteinase K 1 x 50µl 1 x 100µl
    Stop Buffer 1 x 4ml 1 x 8ml
    TdT Enzyme 1 x 40µl 1 x 70µl
    TdT Equilibration Buffer 1 x 4ml 1 x 8ml
    TdT Labeling Reaction Mix 1 x 1.3ml 2 x 1.3ml
  • Research areas

    • Kits/ Lysates/ Other
    • Kits
    • Molecular Biology Kits
    • DNA Isolation Kits
    • Kits/ Lysates/ Other
    • Kits
    • Apoptosis Kits
    • DNA Fragmentation
    • DNA ladder
  • Relevance

    Internucleosomal DNA fragmentation is a hallmark of apoptosis in mammalian cells.

Associated products

  • Assay kits

    • TUNEL Assay Kit - FITC (ab66108)
    • TUNEL Assay Kit - BrdU-Red (ab66110)
  • Related Products

    • PAP pen (ab2601)
    • Mounting Medium for IHC - Aqueous (ab64230)

Images

  • TUNEL assay staining
    TUNEL assay stainingGao Y et al. Reproductive Biology and Endocrinology 15:94 (2017) Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

    Gao Y et al used In situ Apoptosis Detection Kit / TUNEL assay ab206386 to analyze tissue sections from mouse ovaries.

    a. Section treated with DNase I as positive control

    b. Negative control without TdT enzyme

    c and f. representative experimental images.

    Nuclei stained with the TUNEL assay are brown. Sections were counter-stained with Methyl Green.

  • ab206386
    ab206386

    Using paraffin fixed human tonsil tissue,10 μm sections (1000X). A] Section processed and counter-stained with methyl green according to the manual. B] Counter-stain step was eliminated to more clearly illustrate the level of positive staining in the germinal centres of tonsil tissue. C] Section treated with DNase I in order to generate a positive control slide. Note all nuclei stain positive. The use of DNase I generates free 3’-OH groups on cellular DNA, these free 3’-OH groups are then labelled with biotin-nucleotide by the TdT in the kit. D] Negative control, the TdT enzyme step was eliminated thereby generating a negative slide.

  • ab206386
    ab206386

    Using paraffin fixed human tonsil tissue, 10 μm sections (1000X)

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (83)

Publishing research using ab206386? Please let us know so that we can cite the reference in this datasheet.

ab206386 has been referenced in 83 publications.

  • Li Z  et al. Vaccine delivery alerts innate immune systems for more immunogenic vaccination. JCI Insight 6:N/A (2021). PubMed: 33690222
  • Wang SH  et al. Insulin-like growth factor binding protein 3 promotes radiosensitivity of oral squamous cell carcinoma cells via positive feedback on NF-?B/IL-6/ROS signaling. J Exp Clin Cancer Res 40:95 (2021). PubMed: 33712045
  • Lin L  et al. A circular RNA derived from PLXNB2 as a valuable predictor of the prognosis of patients with acute myeloid leukaemia. J Transl Med 19:123 (2021). PubMed: 33757550
  • Lagnado A  et al. Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner. EMBO J 40:e106048 (2021). PubMed: 33764576
  • McKelvey KJ  et al. Glycolysis and Fatty Acid Oxidation Inhibition Improves Survival in Glioblastoma. Front Oncol 11:633210 (2021). PubMed: 33854970
View all Publications for this product

Customer reviews and Q&As

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1-2 of 2 Abreviews or Q&A

Not as straightforward as I'd hoped

Average Average 3/5 (Ease of Use)
Abreviews
Abreviews
abreview image
Bought this kit hoping the staining would be relatively straightforward. The instructions are pretty easy to follow but the results have left something to be desired. I've done the staining twice now and had the same result both times. The samples, negative control, and positive control all have about the same amount of staining. It's pretty difficult to distinguish any positive cells from the background. I think I will need to optimize the proteinase K incubation (possibly doing heat + citrate buffer instead) and / or the amount that the samples are fixed. Overall, using this kit hasn't been as easy as I'd hoped for.
I'm using 4um fixed paraffin embedded mouse brain tissue.

Abcam user community

Verified customer

Submitted Jul 31 2021

TUNEL Assay Kit-HRP-DAB used on human tonsil paraffin sections

Average Average 3/5 (Ease of Use)
Abreviews
Abreviews
abreview image
Used on 4 µm paraffin sections human tonsil following instructions from the kit.

Abcam user community

Verified customer

Submitted Oct 12 2019

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