S7110
ApopTag Fluorescein In Situ Apoptosis Detection Kit
The ApopTag Fluorescein In Situ Apoptosis Detection Kit detects apoptotic cells in situ by the indirect TUNEL method, utilizing an anti-digoxigenin antibody that is conjugated to a Fluorescein reporter molecule.
Synonym(s):
Apoptosis detection kit
Sign Into View Organizational & Contract Pricing
All Photos(1)
About This Item
UNSPSC Code:
12161503
eCl@ss:
32161000
NACRES:
NA.84
Recommended Products
Quality Level
manufacturer/tradename
ApopTag
Chemicon®
technique(s)
flow cytometry: suitable
immunocytochemistry: suitable
immunohistochemistry (formalin-fixed, paraffin-embedded sections): suitable
detection method
fluorometric
shipped in
dry ice
General description
The ApopTag Fluorescein In Situ Apoptosis Detection Kit detects apoptotic cells in situ by the indirect TUNEL method, utilizing an anti-digoxigenin antibody that is conjugated to a Fluorescein reporter molecule. It provides indirect immunofluorescence staining for 40 samples. Results are analyzed using either flow cytometry or fluorescence microscopy.
The ApopTag Fluorescein In Situ Apoptosis Detection Kit has been tested for specific staining in these model systems: (a) human normal peripheral blood lymphocytes induced with dexamethasone as stained in cytospins, (b) rat regressing mammary gland as stained in formalin-fixed, paraffin-embedded sections, and (c) human leukemic peripheral blood lymphocytes induced with camptothecin, as stained in cell suspensions and used for quantitative flow cytometry.
The ApopTag Fluorescein In Situ Apoptosis Detection Kit has been tested for specific staining in these model systems: (a) human normal peripheral blood lymphocytes induced with dexamethasone as stained in cytospins, (b) rat regressing mammary gland as stained in formalin-fixed, paraffin-embedded sections, and (c) human leukemic peripheral blood lymphocytes induced with camptothecin, as stained in cell suspensions and used for quantitative flow cytometry.
Application
INTRODUCTION
ApopTag In Situ Apoptosis Detection Kits label apoptotic cells in research samples by modifying genomic DNA utilizing terminal deoxynucleotidyl transferase (TdT) for detection of positive cells by specific staining. This manual contains information and protocols for the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Catalog number S7110).
Principles of the Procedure
The reagents provided in all ApopTag Kits are designed to label the free 3′OH DNA termini in situ with chemically labeled and unlabeled nucleotides. The nucleotides contained in the Reaction Buffer are enzymatically added to the DNA by terminal deoxynucleotidyl transferase (TdT) (13, 31). TdT catalyzes a template-independent addition of nucleotide triphosphates to the 3′-OH ends of double-stranded or single-stranded DNA. The incorporated nucleotides form an oligomer composed of digoxigenin nucleotide and unlabeled nucleotide in a random sequence. The ratio of labeled to unlabeled nucleotide in ApopTag Kits is optimized to promote anti-digoxigenin antibody binding, or to minimize fluorescein self-quenching. The exact length of the oligomer added has not been measured.
DNA fragments which have been labeled with the digoxigenin-nucleotide are then allowed to bind an anti-digoxigenin antibody that is conjugated to fluorescein (Figure 1A). Fluorescent antibodies provide sensitive detection in immunohistochemistry or immunocytochemistry (i.e. on tissue or cells) and are not subject to experimental variations due to the substrate or the development step. This mixed molecular biological-histochemical systems allows for sensitive and specific staining of very high concentrations of 3′-OH ends that are localized in apoptotic bodies.The ApopTag system differs significantly from previously described in situ labeling techniques for apoptosis (13, 16, 38, 46), in which avidin binding to cellular biotin can be a source of error. The digoxigenin/anti-digoxigenin system has been found to be equally sensitive to avidin/biotin systems (22). Immunochemically-similar ligands for binding of the anti-digoxigenin antibody are generally insignificant in animal tissues, ensuring low background staining. Affinity purified sheep polyclonal antibody is the specific anti-digoxigenin reagent used in ApopTag Kits and exhibits <1% cross-reactivity with the major vertebrate steroids. In addition, the Fc portion of this antibody has been removed by proteolytic digestion to eliminate any non-specific adsorption to cellular Fc receptors.
ApopTag In Situ Apoptosis Detection Kits label apoptotic cells in research samples by modifying genomic DNA utilizing terminal deoxynucleotidyl transferase (TdT) for detection of positive cells by specific staining. This manual contains information and protocols for the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Catalog number S7110).
Principles of the Procedure
The reagents provided in all ApopTag Kits are designed to label the free 3′OH DNA termini in situ with chemically labeled and unlabeled nucleotides. The nucleotides contained in the Reaction Buffer are enzymatically added to the DNA by terminal deoxynucleotidyl transferase (TdT) (13, 31). TdT catalyzes a template-independent addition of nucleotide triphosphates to the 3′-OH ends of double-stranded or single-stranded DNA. The incorporated nucleotides form an oligomer composed of digoxigenin nucleotide and unlabeled nucleotide in a random sequence. The ratio of labeled to unlabeled nucleotide in ApopTag Kits is optimized to promote anti-digoxigenin antibody binding, or to minimize fluorescein self-quenching. The exact length of the oligomer added has not been measured.
DNA fragments which have been labeled with the digoxigenin-nucleotide are then allowed to bind an anti-digoxigenin antibody that is conjugated to fluorescein (Figure 1A). Fluorescent antibodies provide sensitive detection in immunohistochemistry or immunocytochemistry (i.e. on tissue or cells) and are not subject to experimental variations due to the substrate or the development step. This mixed molecular biological-histochemical systems allows for sensitive and specific staining of very high concentrations of 3′-OH ends that are localized in apoptotic bodies.The ApopTag system differs significantly from previously described in situ labeling techniques for apoptosis (13, 16, 38, 46), in which avidin binding to cellular biotin can be a source of error. The digoxigenin/anti-digoxigenin system has been found to be equally sensitive to avidin/biotin systems (22). Immunochemically-similar ligands for binding of the anti-digoxigenin antibody are generally insignificant in animal tissues, ensuring low background staining. Affinity purified sheep polyclonal antibody is the specific anti-digoxigenin reagent used in ApopTag Kits and exhibits <1% cross-reactivity with the major vertebrate steroids. In addition, the Fc portion of this antibody has been removed by proteolytic digestion to eliminate any non-specific adsorption to cellular Fc receptors.
The ApopTag Fluorescein In Situ Apoptosis Detection Kit detects apoptotic cells in situ by the indirect TUNEL method, utilizing an anti-digoxigenin antibody that is conjugated to a Fluorescein reporter molecule.
Components
Equilibration Buffer 90416 3.0 mL -15°C to -25°C
Reaction Buffer 90417 2.0 mL -15°C to -25°C
TdT Enzyme 90418 0.64 mL -15°C to -25°C
Stop/Wash Buffer 90419 20 mL -15°C to -25°C
Blocking Solution 90425 2.6 mL -15°C to -25°C
Anti-Digoxigenin-Fluorescein* 90426 2.1 mL 2°C to 8°C
Plastic Coverslips 90421 100 ea. Room Temp.
*affinity purified sheep polyclonal antibody
Reaction Buffer 90417 2.0 mL -15°C to -25°C
TdT Enzyme 90418 0.64 mL -15°C to -25°C
Stop/Wash Buffer 90419 20 mL -15°C to -25°C
Blocking Solution 90425 2.6 mL -15°C to -25°C
Anti-Digoxigenin-Fluorescein* 90426 2.1 mL 2°C to 8°C
Plastic Coverslips 90421 100 ea. Room Temp.
*affinity purified sheep polyclonal antibody
Storage and Stability
1. Store the kit at -15°C to -25°C until the first use. After the first use, if the kit will be used within three months, store the TdT Enzyme (#90418) at -15°C to -25°C and store the remaining components at 2°C to 8°C.
2. Protect the anti-digoxigenin fluorescein antibody (#90426) from unnecessary exposure to light.
Precautions
1. The following kit components contain potassium cacodylate (dimethylarsinic acid) as a buffer: Equilibration Buffer (#90416), Reaction Buffer (#90417), and TdT Enzyme (#90418). These components are harmful if swallowed; avoid contact with skin and eyes (wear gloves, glasses) and wash areas of contact immediately.
2. Antibody Conjugates (#90426) and Blocking Solutions (#90425) contain 0.08% sodium azide as a preservative.
3. TdT Enzyme (#90418) contains glycerol and will not freeze at -20°C. For maximum shelf life, do not warm this reagent to room temperature before dispensing.
2. Protect the anti-digoxigenin fluorescein antibody (#90426) from unnecessary exposure to light.
Precautions
1. The following kit components contain potassium cacodylate (dimethylarsinic acid) as a buffer: Equilibration Buffer (#90416), Reaction Buffer (#90417), and TdT Enzyme (#90418). These components are harmful if swallowed; avoid contact with skin and eyes (wear gloves, glasses) and wash areas of contact immediately.
2. Antibody Conjugates (#90426) and Blocking Solutions (#90425) contain 0.08% sodium azide as a preservative.
3. TdT Enzyme (#90418) contains glycerol and will not freeze at -20°C. For maximum shelf life, do not warm this reagent to room temperature before dispensing.
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Signal Word
Danger
Hazard Statements
Precautionary Statements
Hazard Classifications
Aquatic Chronic 2 - Carc. 1B - STOT RE 2 Inhalation
Target Organs
Respiratory Tract
Storage Class Code
6.1C - Combustible acute toxic Cat.3 / toxic compounds or compounds which causing chronic effects
Certificates of Analysis (COA)
Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.
Already Own This Product?
Find documentation for the products that you have recently purchased in the Document Library.
Customers Also Viewed
Mary Truscott et al.
The Journal of biological chemistry, 282(41), 30216-30226 (2007-08-08)
Proteolytic processing at the end of the G(1) phase generates a CUX1 isoform, p110, which functions either as a transcriptional activator or repressor and can accelerate entry into S phase. Here we describe a second proteolytic event that generates an
Tsutomu Sasaki et al.
Stroke, 38(5), 1597-1605 (2007-03-24)
Brain ischemia stimulates neurogenesis. However, newborn neurons show a progressive decrease in number over time. Under normal conditions, the cAMP-cAMP responsive element binding protein (CREB) pathway regulates the survival of newborn neurons. Constitutive activation of CREB after brain ischemia also
Kirsten A Baken et al.
Methods (San Diego, Calif.), 41(1), 132-141 (2006-12-13)
Microarray analysis is used for simultaneous measurement of expression of thousands of genes in a given sample and as such extends and deepens our understanding of biological processes. Application of the technique in toxicology is referred to as toxicogenomics. The
Louis Tong et al.
Investigative ophthalmology & visual science, 47(10), 4295-4301 (2006-09-28)
Ultraviolet light (UVB) is known to cause apoptosis in human corneal epithelial cells. This study evaluates the role of transglutaminase in regulating tumor necrosis factor (TNF) receptor clustering as well as caspase activation in UVB-induced apoptosis in human corneal epithelial
Konstantinos Ampatzis et al.
The European journal of neuroscience, 25(4), 1030-1040 (2007-03-03)
It has been reported that neurons generated in the adult brain show sex-specific differences in several brain regions of lower vertebrates and mammals. The present study questioned whether cell proliferation and survival in the adult zebrafish (Danio rerio) cerebellum, the
Articles
Cellular apoptosis assays to detect programmed cell death using Annexin V, Caspase and TUNEL DNA fragmentation assays.
Our team of scientists has experience in all areas of research including Life Science, Material Science, Chemical Synthesis, Chromatography, Analytical and many others.
Contact Technical Service