S7101 ApopTag® Plus Peroxidase In Situ Apoptosis Kit

S7101
40 assays  
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Chromogenic
      Description
      Catalogue NumberS7101
      Brand Family Chemicon®
      Trade Name
      • ApopTag
      • Chemicon
      DescriptionApopTag® Plus Peroxidase In Situ Apoptosis Kit
      OverviewThe ApopTag® Plus Peroxidase In Situ Apoptosis Detection Kit detects apoptotic cells by labeling and detecting DNA strand breaks by the indirect TUNEL method. The kit provides sufficient reagents for immunoperoxidase staining of 40 samples, and includes ApopTag(R) Positive control Slides and DAB buffer and substrate. Results are visualized using brightfield microscopy.

      The ApopTag® Peroxidase Kits have been qualified for use in histochemical and cytochemical staining of the following specimens: formalin-fixed, paraffin-embedded tissues, cryostat sections, cell suspensions, cytospins, and cell cultures. Whole mount-methods have been developed (34, 45). (See datasheet Sec. V. for References).

      The staining specificity of the ApopTag® Peroxidase Kits has been demonstrated by Chemicon and many other laboratories. Chemicon has tested many types of model cell and tissue systems, including: (a) human prostate, thymus, and large intestine (in-house data); (b) rat ventral prostate post-castration (21), (c) rat thymus lymphocytes treated in vitro with dexamethasone (3, 13), (d) 14-day mouse embryo limbs (1) and (e) rat mammary gland in regression after weaning (36). In the thymocyte and prostate models, agarose gel electrophoresis was used to assess the amount of DNA laddering, which peaked coincidentally with the maximum percentage of stained cells. Numerous journal publications from laboratories worldwide have established the usefulness of ApopTag® Kits. (See datasheet Sec. V. References, Publications Citing ApopTag® Kits).
      Background InformationApoptosis is a form of cell death that eliminates compromised or superfluous cells. It is controlled by multiple signaling and effector pathways that mediate active responses to external growth, survival, or death factors. Cell cycle checkpoint controls are linked to apoptotic enzyme cascades, and the integrity of these and other links can be genetically compromised in many diseases, such as cancer. There are many books in print and hundreds of recent review articles about all aspects of apoptosis (e.g. 7, 11, 19, 24, 39, 42) and the methods for detecting it (e.g. 10, 32, 36).

      Of all the aspects of apoptosis, the defining characteristic is a complete change in cellular morphology. As observed by electron microscopy, the cell undergoes shrinkage, chromatin margination, membrane blebbing, nuclear condensation and then segmentation, and division into apoptotic bodies which may be phagocytosed (11, 19, 24). The characteristic apoptotic bodies are short-lived and minute, and can resemble other cellular constituents when viewed by brightfield microscopy. DNA fragmentation in apoptotic cells is followed by cell death and removal from the tissue, usually within several hours (7). A rate of tissue regression as rapid as 25% per day can result from apparent apoptosis in only 2-3% of the cells at any one time (6). Thus, the quantitative measurement of an apoptotic index by morphology alone can be difficult.

      DNA fragmentation is usually associated with ultrastructural changes in cellular morphology in apoptosis (26, 38). In a number of well-researched model systems, large fragments of 300 kb and 50 kb are first produced by endonucleolytic degradation of higher-order chromatin structural organization. These large DNA fragments are visible on pulsed-field electrophoresis gels (5, 43, 44). In most models, the activation of Ca2+- and Mg2+-dependent endonuclease activity further shortens the fragments by cleaving the DNA at linker sites between nucleosomes (3). The ultimate DNA fragments are multimers of about 180 bp nucleosomal units. These multimers appear as the familiar "DNA ladder" seen on standard agarose electrophoresis gels of DNA extracted from many kinds of apoptotic cells (e.g. 3, 7,13, 35, 44).

      Another method for examining apoptosis via DNA fragmentation is by the TUNEL assay, (13) which is the basis of ApopTag® technology. The DNA strand breaks are detected by enzymatically labeling the free 3'-OH termini with modified nucleotides. These new DNA ends that are generated upon DNA fragmentation are typically localized in morphologically identifiable nuclei and apoptotic bodies. In contrast, normal or proliferative nuclei, which have relatively insignificant numbers of DNA 3'-OH ends, usually do not stain with the kit. ApopTag® Kits detect single-stranded (25) and double-stranded breaks associated with apoptosis. Drug-induced DNA damage is not identified by the TUNEL assay unless it is coupled to the apoptotic response (8). In addition, this technique can detect early-stage apoptosis in systems where chromatin condensation has begun and strand breaks are fewer, even before the nucleus undergoes major morphological changes (4, 8).

      Apoptosis is distinct from accidental cell death (necrosis). Numerous morphological and biochemical differences that distinguish apoptotic from necrotic cell death are summarized in the following table (adapted with permission from reference 39). ApopTag® In Situ Apoptosis Detection Kits distinguish apoptosis from necrosis by specifically detecting DNA cleavage and chromatin condensation associated with apoptosis. However, there may be some instances where cells exhibiting necrotic morphology may stain lightly (14, 29) or, in rare instances, DNA fragmentation can be absent or incomplete in induced apoptosis (11). It is, therefore, important to evaluate ApopTag® staining results in conjunction with morphological criteria. Visualization of positive ApopTag® results should reveal focal in situ staining inside early apoptotic nuclei and apoptotic bodies. This positive staining directly correlates with the more typical biochemical and morphological aspects of apoptosis.

      Since an understanding of cell morphology is critical for data interpretation and because of the potential for experimentally modifying or overcoming normal apoptotic controls, the following strategy is advised. When researching a new system, the staging and correlation of apoptotic morphology and DNA fragmentation should be characterized. In some tissues, cytoplasmic shrinkage may be indicated by a clear space surrounding the cell. The nuclear morphology of positive cells should be carefully observed at high magnification (400x-1000x). Early staged positive, round nuclei may have observable chromatin margination. Condensed nuclei of middle stages, and apoptotic bodies, usually are stained. Apoptotic bodies may be found either in the extracellular space or inside of phagocytic cells. It is highly recommended that less experienced observers should refer to illustrations of dying cells for comparison with new data (e.g. 11, 19, 24).

      An additional, although far less sensitive, method of confirming ApopTag® staining results is the detection of DNA fragmentation on agarose gels. If a large percent of the cells in the tissue are apoptotic, then electrophoresis of extracted total genomic DNA and standard dye staining can be used to corroborate the in situ staining. However, the single-cell sensitivity of ApopTag® histochemistry is far higher than this method. DNA laddering data of comparable sensitivity may be obtained in several other ways. These include methods for selectively extracting the low molecular weight DNA (15), for preparing radiolabeled DNA (30, 40) in combination with resin-bed purification of DNA (12), and for DNA amplification by PCR (35).

      The in situ staining of DNA strand breaks detected by the TUNEL assay and subsequent visualization by light microscopy gives biologically significant data about apoptotic cells which may be a small percentage of the total population (13, 16). Apoptotic cells stained positive with ApopTag® Kits are easier to detect and their identification is more certain, as compared to the examination of simply histochemically stained tissues.
      Materials Required but Not DeliveredSolvents and Media

      a. Deionized water (dH2O)

      b. Xylene

      c. Ethanol: absolute, 95%, 70%, diluted in dH2O water

      d. 100% n-butanol (1-butanol)

      e. Ethanol: acetic acid, 2:1 (v:v) (for tissue cryosection or cells protocols)

      f. Slide mounting medium (Permount or equivalent for glass support, Aquamount or equivalent for plastic embedding or support). See Sec. IV. Appendix, TECH NOTE #15: Fixation using plastic supports.

      Solutions

      Note: See Sec. IV. Appendix: Reagent Preparation for specific instructions for preparing these solutions.

      a. 1% paraformaldehyde in PBS, pH 7.4 (methanol-free formaldehyde for tissue cryosections or cells). See Sec. IV. Appendix, TECH NOTE #2: Fixatives and fixation.

      b. 10% (v:v) neutral buffered formalin (for fixation before paraffin-embedding). See Sec. IV. Appendix, TECH NOTE #2: Fixatives and fixation.

      c. PBS (50 mM sodium phosphate, pH 7.4, 200 mM NaCl)

      d. Hydrogen peroxide, commercial 30% solution

      e. Protein Digesting Enzyme or proteinase K (for paraffin-embedded tissue protocol).

      f. 0.5% (w:v) methyl green, free of crystal violet

      g. Triton X-100 10% (w:v) stock solution (optional)

      h. 10 mM citrate buffer, pH 6.0 (optional)

      Materials

      a. Silanized glass slides

      b. Glass coverslips (for oil immersion objective, use 22 x 50 mm)

      c. Adjustable micropipettors

      d. Glass or plastic coplin jars

      e. Forceps for handling plastic coverslips (optional)

      f. Humidified chamber. See Sec. IV. Appendix, TECH NOTE #7: Containers.

      g. Microcentrifuge tubes

      Equipment

      a. 37°C covered water bath or incubator at 37°C

      b. Light microscope equipped with brightfield optics (40x and 100x objectives)
      References
      Product Information
      Components
      • Equilibration Buffer 90416 3.0 mL -15°C to -25°C
      • Reaction Buffer 91417 2.0 mL -15°C to -25°C
      • TdT Enzyme 90418 0.672 mL -15°C to -25°C
      • Stop/Wash Buffer 90419 20 mL -15°C to -25°C
      • Anti-Digoxigenin-Peroxidase* 90420 3.0 mL 2°C to 8°C
      • Plastic Coverslips 90421 100 ea. Room Temp.
      • Control Slides 90422 2 each Room Temp.
      • DAB Substrate 90423 130 μL 2°C to 8°C
      • DAB Dilution Buffer 90424 6.5 mL 2°C to 8°C
      • *affinity purified sheep polyclonal antibody
      • Number of samples per kit: Sufficient materials are provided to stain 40 tissue specimens of approximately 5 cm2 each when used according to instructions. Reaction Buffer will be fully consumed before other reagents when kits are used for slide-mounted specimens.
      Detection methodChromogenic
      Applications
      ApplicationThe ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit detects apoptotic cells by labeling & detecting DNA strand breaks by the indirect TUNEL method.
      Application NotesINTRODUCTION

      ApopTag® In Situ Apoptosis Detection Kits label apoptotic cells in research samples by modifying DNA fragments utilizing terminal deoxynucleotidyl transferase (TdT) for detection of apoptotic cells by specific staining.

      This manual contains information and protocols for the ApopTag® Plus In Situ Apoptosis Detection Kit.

      Principles of the Procedure

      The reagents provided in ApopTag® Peroxidase 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-conjugated nucleotide and unlabeled nucleotide in a random sequence. The ratio of labeled to unlabeled nucleotide in ApopTag® Peroxidase Kits is optimized to promote anti-digoxigenin antibody binding. 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 a peroxidase reporter molecule (Figure 1A). The bound peroxidase antibody conjugate enzymatically generates a permanent, intense, localized stain from chromogenic substrates, providing sensitive detection in immunohistochemistry or immunocytochemistry (i.e. on tissue or cells). 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). The sole natural source of digoxigenin is the digitalis plant. 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. This antibody 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.

      Results using ApopTag® Kits have been widely published (see Sec. V. References, Publications Citing ApopTag® Kits). The ApopTag® product line provides various options in experimental design. A researcher can choose to detect staining by brightfield or fluorescence microscopy or by flow cytometry, depending on available expertise and equipment. There are also opportunities to study other proteins of interest in the context of apoptosis when using ApopTag® Kits. By using antibodies conjugated with an enzyme other than peroxidase and an appropriate choice of substrate, it is possible to simultaneously examine another protein and apoptosis using ApopTag® Peroxidase Kits.
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • 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.
      Storage and Shipping Information
      Storage ConditionsStore 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.

      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. DAB (3,3' diaminobenzidine) Substrate (90423) has been demonstrated to be a potential carcinogen and skin contact should be avoided. If skin contact does occur, flush with copious amounts of dH2O.

      3. Antibody Conjugates (90420) contains 0.08% sodium azide as a preservative.

      4. TdT Enzyme (90418) contains glycerol and will not freeze at -20°C. For maximum shelf life, do not warm this reagent to room temp. before dispensing.
      Packaging Information
      Material Size40 assays
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      SDS

      Title

      Safety Data Sheet (SDS) 

      References

      Reference overviewPub Med ID
      Exposure to heavy ion radiation induces persistent oxidative stress in mouse intestine.
      Kamal Datta,Shubhankar Suman,Bhaskar V S Kallakury,Albert J Fornace
      PloS one  7  2012

      Show Abstract
      22936983 22936983
      Compartmentalization and regulation of iron metabolism proteins protect male germ cells from iron overload.
      Yael Leichtmann-Bardoogo,Lyora A Cohen,Avital Weiss,Britta Marohn,Stephanie Schubert,Andreas Meinhardt,Esther G Meyron-Holtz
      American journal of physiology. Endocrinology and metabolism  302  2012

      Show Abstract
      22496346 22496346
      Effects of resveratrol on blood homocysteine level, on homocysteine induced oxidative stress, apoptosis and cognitive dysfunctions in rats.
      Sema Tulay Koz,Ebru Onalan Etem,Gıyasettin Baydas,Huseyin Yuce,Halil Ibrahim Ozercan,Tuncay Kuloğlu,Suleyman Koz,Arzu Etem,Nevgul Demir
      Brain research  1484  2012

      Show Abstract
      22995369 22995369
      Biomarkers of phenethyl isothiocyanate-mediated mammary cancer chemoprevention in a clinically relevant mouse model.
      Shivendra V Singh,Su-Hyeong Kim,Anuradha Sehrawat,Julie A Arlotti,Eun-Ryeong Hahm,Kozue Sakao,Jan H Beumer,Rachel C Jankowitz,Kumar Chandra-Kuntal,Joomin Lee,Anna A Powolny,Rajiv Dhir
      Journal of the National Cancer Institute  104  2012

      Show Abstract
      22859850 22859850
      SMARCAL1 deficiency predisposes to non-Hodgkin lymphoma and hypersensitivity to genotoxic agents in vivo.
      Alireza Baradaran-Heravi,Anja Raams,Joanna Lubieniecka,Kyoung Sang Cho,Kristi A Dehaai,Mitra Basiratnia,Pierre-Olivier Mari,Yutong Xue,Michael Rauth,Ann Haskins Olney,Mary Shago,Kunho Choi,Rosanna A Weksberg,Malgorzata J M Nowaczyk,Weidong Wang,Nicolaas G J Jaspers,Cornelius F Boerkoel
      American journal of medical genetics. Part A  158A  2012

      Show Abstract
      22888040 22888040
      Effect of a Low-Fat Diet Combined with IGF-1 Receptor Blockade on 22Rv1 Prostate Cancer Xenografts.
      Ramdev Konijeti,Satomi Koyama,Ashley Gray,R James Barnard,Jonathan W Said,Brandon Castor,David Elashoff,Junxiang Wan,Pedro J Beltran,Frank J Calzone,Pinchas Cohen,Colette Galet,William J Aronson
      Molecular cancer therapeutics  11  2012

      Show Abstract
      22562985 22562985
      Early regression of the dental lamina underlies the development of diphyodont dentitions.
      M Buchtová,J Stembírek,K Glocová,E Matalová,A S Tucker
      Journal of dental research  91  2012

      Show Abstract
      22442052 22442052
      Effects of prolonged water washing of tissue samples fixed in formalin on histological staining.
      Suzuki, Y; Imada, T; Yamaguchi, I; Yoshitake, H; Sanada, H; Kashiwagi, T; Takaba, K
      Biotechnic & histochemistry : official publication of the Biological Stain Commission  87  241-8  2012

      Show Abstract
      21958122 21958122
      Evaluation of apoptosis along with BCL-2 and Ki-67 expression in patients with intestinal metaplasia.
      Gulbanu Erkan,Ipek Isik Gonul,Ugur Kandilci,Ayse Dursun
      Pathology, research and practice  208  2012

      Show Abstract
      22277792 22277792
      Does furan affect the thymus in growing male rats?
      E Arzu Koçkaya,Aysun Kılıç,Elif Karacaoğlu,Güldeniz Selmanoğlu
      Drug and chemical toxicology  35  2012

      Show Abstract
      22289615 22289615

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      ApopTag® Plus Peroxidase In Situ Apoptosis Detection Kit

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