ApopTag Red In Situ Apoptosis Detection Kit

The ApopTag Red In Situ Apoptosis Detection Kit detects apoptosis in situ by the indirect TUNEL method, utilizing an anti-digoxigenin antibody with a rhodamine fluorochrome. The kit provides indirect immunofluorescence staining for 40 samples. Results are analyzed using either fluorescence microscopy or flow cytometry (requires a special laser and filters for rhodamine).

Indirect ApopTag Kits (S7100, S7101, S7110, S7111 and S7165) 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).

ApopTag Peroxidase Kits staining specificity 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).

ApopTag Fluorescein and Rhodamine Kits have 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 (22).

In some instances, certain tissues contain cell types which bind any fluorescein labeled nucleotide such as that contained in the ApopTag Fluorescein Direct Kit (S7160). It is therefore recommended that this kit (S7160) be used to stain cell suspensions, cytospins and cell cultures, but not tissue sections. Chemicon suggests the use of the ApopTag Fluorescein Kits (S7110, S7111) or the ApopTag Red Kit for staining tissue sections. See datasheet for references.

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 Red Apoptosis Detection Kit (S7165).

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 or fluorescein 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.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. There is also a choice of fluorophores (fluorescein and rhodamine) using ApopTag technology. Flexibility exists in choosing antibody-fluor combinations to study other important proteins.

The ApopTag Red In Situ Apoptosis Detection Kit detects apoptosis in situ by the indirect TUNEL method, utilizing an anti-digoxigenin antibody with a rhodamine fluorochrome.

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-Rhodamine* 90429 2.1 mL 2°C to 8°C
Plastic Coverslips 90421 100 ea. Room Temp.
*affinity purified sheep polyclonal antibody
Number of tests 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.

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 rhodamine antibody (90429) 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 Conjugate (90429) and Blocking Solution (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 temp. before dispensing.

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