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  • : S7100
  • : 40 assays
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Detection of apoptotic cells in human lymph node with ApopTag® Peroxidase. The tissue was fixed in 10% neutral-buffered formalin and paraffin-embedded...

ApopTag® Peroxidase In Situ Apoptosis Detection Kit {40 tests}

ApopTag® Peroxidase In Situ Apoptosis Detection Kit

Description:
ApopTag® Peroxidase In Situ Apoptosis Detection Kit
Trade Name:
  • ApopTag
  • Chemicon (Millipore)
Qty/Pk:
40 assays
Product Overview:
The ApopTag® Peroxidase In Situ Apoptosis Detection Kit detects apoptotic cells in situ by labeling and detecting DNA strand breaks by the TUNEL method. The kit provides sufficient reagents for immunoperoxidase staining of 40 samples. Results are visualized using brightfield microscopy.
Background Information:
Apoptosis 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).
Applications:
The ApopTag Peroxidase In Situ Apoptosis Detection Kit detects apoptotic cells in situ by labeling & detecting DNA strand breaks by the TUNEL method.
Application Notes:
INTRODUCTION

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.

The ApopTag® Peroxidase Kit has 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).

The staining specificity of the ApopTag® Peroxidase Kit 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 Sec. V. References, Publications Citing ApopTag® Kits).

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.
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.
Components:
  • Equilibration Buffer: 3.0 mL -15°C to -25°C
  • Reaction Buffer 2.0 mL -15°C to -25°C
  • TdT Enzyme 0.64 mL -15°C to -25°C
  • Stop/Wash Buffer 20 mL -15°C to -25°C
  • Anti-Digoxigenin-Peroxidase* 3.0 mL 2°C to 8°C
  • Plastic Coverslips 100 ea. Room Temp.
  • Note: Separate purchase of DAB (Peroxidase Substrate) is required. It is not supplied with this kit.
  • 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.
Brand Family:
Chemicon
Storage Conditions:
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.

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 (90420) and Blocking Solutions (#10 and #13) 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 temp. before dispensing.
Detection Methods:
Chromogenic
Product Name:
ApopTag® Peroxidase In Situ Apoptosis Detection Kit
Materials Required but Not Delivered:
Solvents and Media
Deionized water (dH2O)

Xylene

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

100% n-butanol (1-butanol)

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

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.

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.

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

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

Hydrogen peroxide, commercial 30% solution

Diaminobenzidine (DAB) in staining buffer (PBS, Tris or other staining buffer recommended by manufacturer). Minimize exposure to heat and light.

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

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

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

10 mM citrate buffer, pH 6.0 (optional)

Materials
Silanized glass slides

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

Adjustable micropipettors

Glass or plastic coplin jars

Forceps for handling plastic coverslips (optional)

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

Microcentrifuge tubes

Equipment
37°C covered water bath or incubator at 37°C

Light microscope equipped with brightfield optics (40x and 100x objectives)

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