ApopTagペルオキシダーゼIn Situアポトーシス検出キット

ApopTagペルオキシダーゼIn Situアポトーシス検出キットは、DNA鎖切断を標識してTUNEL法で検出することにより、アポトーシス細胞をin situで検出します。本キットには、40サンプルの免疫ペルオキシダーゼ染色に十分な試薬が含まれています。結果は明視野顕微鏡を用いて可視化されます。

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).

平衡バッファー3.0 mL -15°C～-25°C

反応バッファー 2.0 mL -15°C～-25°C

TdT酵素 0.64 mL -15°C～-25°C

停止／洗浄バッファー 20 mL -15°C～-25°C

抗ジゴキシゲニン-ペルオキシダーゼ* 3.0 mL 2°C～8°C

プラスチックカバースリップ 100 ea.室温。

注記：DAB（ペルオキシダーゼ基質）は別途購入する必要があります。このキットには付属していません。

1キットあたりのサンプル数：指示に従って使用する場合は、約5 mm²の組織サンプル40点分の染色に十分な材料が含まれています。キットをスライド標本に使用する場合は、他の試薬より先に反応バッファーが完全に消費されます。

最初に使用するまでは、キットを-15°C～-25°Cで保存してください。最初に使用した後、キットを3か月以内に使用する場合は、TdT酵素（90418）は-15°C～-25°Cで保存し、その他の構成要素は2°C～8°Cで保存してください。

使用上の注意

1. 以下のキット構成要素には、バッファーとしてカコジル酸カリウム（ジメチルアルシン酸）が含まれています：平衡バッファー（#90416）、反応バッファー（#90417）、TdT酵素（#90418）。これらの構成要素は飲み込むと有害です。皮膚と眼への接触を避け（手袋、眼鏡を着用）、接触した部位はすぐに洗ってください。

2.抗体コンジュゲート（90420）およびブロッキング溶液（10および13）には、保存剤として0.08%アジ化ナトリウムが含まれています。

3.TdT酵素（#90418）にはグリセロールが含まれており、-20°Cでは凍りません。有効期間を最長にするため、分注する前にこの試薬が室温まで温まらないようにしてください。

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