ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit

The 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.

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 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.

The 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.

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.

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

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