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S7111 | ApopTag® Plus In Situ Apoptosis Fluorescein Detection Kit

S7111
40 assays  
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      Tabela com principais espec.

      Key ApplicationsDetection Methods
      ICC, IHC, IH(P), FC Fluorescent
      Description
      Catalogue NumberS7111
      Brand Family Chemicon®
      Trade Name
      • ApopTag
      • Chemicon
      DescriptionApopTag® Plus In Situ Apoptosis Fluorescein Detection Kit
      OverviewThe ApopTag® Plus 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. The kit provides sufficient reagents for immunofluorescence staining of 40 samples and includes ApopTag® Positive Control Slides. 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.
      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 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. Another feature of ApopTag® is that quantitative results can be obtained using flow cytometry, since end-labeling methodology detects apoptotic cells with a >10-fold higher sensitivity than necrotic cells (14,17). In addition, the occurrence of DNA fragmentation with regard to the cell cycle phase of apoptotic cells can be examined using the TUNEL assay and flow cytometry (16,18).
      Materials Required but Not DeliveredSolvents and Media

      a. Deionized water (dH2O)

      b. Xylene

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

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

      e. Slide mounting medium (Antifade)

      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. Protein Digesting Enzyme or proteinase K (for paraffin-embedded tissue protocol). Prepare a 5 mg/mL stock in PBS and store in small frozen aliquots.

      e. 0.5-1.0 μg/mL Propidium Iodide in Antifade (S7112)

      f. 0.5-1.0 μg/mL DAPI (4'-6' diamino-2-phenylindole) in Antifade (S7113)



      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

      g. 37°C covered water bath, or incubator at 37°C

      Equipment

      Light microscope equipped with brightfield optics (40x and 10x objectives) and also equipped for fluorescence. See Sec. IV. Appendix, TECH NOTE #6: Required fluorescence filters.
      References
      Product Information
      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.
      • Control Slides 90422 2 ea. Room Temp.
      • *affinity purified sheep polyclonal antibody
      Detection methodFluorescent
      Applications
      ApplicationThe ApopTag Plus 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.
      Key Applications
      • Immunocytochemistry
      • Immunohistochemistry
      • Immunohistochemistry (Paraffin)
      • Flow Cytometry
      Application NotesINTRODUCTION

      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® Plus Fluorescein In Situ Apoptosis Detection Kit (Catalog number S7111).

      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.
      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 Conditions1. 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.
      Packaging Information
      Material Size40 assays
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      MSDS

      Título

      Ficha de Segurança de Produtos (MSDS) 

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      Brochura

      Título
      Advancing cancer research: From hallmarks & biomarkers to tumor microenvironment progression
      Comprehensive solutions for studying cell health - Life, death, and everything in between.

      Informações técnicas

      Título
      A Comparative Analysis of Human Embryonic Stem Cells Cultured in a Variety of Media Conditions

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