S7100

Kit ApopTag Peroxidase per il rilevamento dell′apoptosi in situ

The ApopTag Peroxidase In Situ Apoptosis Detection Kit detects apoptotic cells in situ by labeling & detecting DNA strand breaks by the TUNEL method.

• Sinonimo/i: ApopTag Kit, In Situ Apoptosis Detection Kit
• Codice UNSPSC: 12161503
• eCl@ss: 32161000
• NACRES: NA.84

Proprietà

• Livello qualitativo: 100
• Produttore/marchio commerciale: ApopTag; Chemicon^®
• Metodo di rivelazione: colorimetric
• Condizioni di spedizione: dry ice

Descrizione

Descrizione generale

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).

Il kit ApopTag di rilevamento dell′apoptosi in situ con perossidasi rileva cellule apoptotiche in situ marcando e rilevando le rotture dei filamenti di DNA con il metodo TUNEL. Il kit fornisce reagenti sufficienti per colorare 40 campioni con immunoperossidasi. Il microscopico in campo chiaro permette di visualizzare i risultati.

Componenti

Tampone di equilibrazione: 3,0 mL tra -15 °C e -25 °C

Tampone di reazione 2,0 mL tra -15 °C e -25 °C

Enzima TdT 0,64 mL tra -15 °C e -25 °C

Tampone di arresto/lavaggio 20 mL tra -15 °C e -25 °C

Anti-digossigenina-perossidasi* 3,0 mL tra 2 °C e 8 °C

Vetrini coprioggetto in plastica 100 per conf. Temp. ambiente

Nota: è necessario acquistare separatamente la DAB (substrato della perossidasi). Non viene fornita con il kit.

Numeri di campioni per kit: i materiali forniti sono sufficienti per colorare 40 campioni di tessuto di circa 5 mm² ciascuno, se usati seguendo le istruzioni. Se si usano i kit per campioni montati su vetrini, il tampone di reazione si esaurisce prima degli altri reagenti.

Stoccaggio e stabilità

Conservare il kit tra -15 °C e -25 °C fino al primo utilizzo. Dopo il primo utilizzo, se il kit sarà usato entro tre mesi, conservare l′Enzima TdT (90418) tra -15 °C e -25 °C e gli altri componenti tra 2 °C e 8 °C.

Precauzioni

1. I seguenti componenti del kit contengono cacodilato di potassio (acido dimetilarsinico) come tampone: Tampone di equilibrazione (90416), Tampone di reazione (90417) ed Enzima TdT (90418). Questi componenti sono nocivi se ingeriti; evitare il contatto con la cute e gli occhi (indossare guanti e occhiali) e lavare immediatamente le superfici di contatto.

2. I coniugati di anticorpi (90420) e le soluzioni di bloccaggio (n. 10 e n. 13) contengono 0,08% di sodio azide come conservante.

3. L′Enzima TdT (90418) contiene glicerolo e non congela a -20 °C. Per ottimizzare la durata di conservazione non scaldare il reagente a temperatura ambiente prima di erogarlo.

Note legali

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Informazioni sulla sicurezza

• Pittogrammi: GHS08,GHS07,GHS09
• Avvertenze: Danger
• Indicazioni di pericolo: H317,H350,H373,H411
• Consigli di prudenza: P202 - P260 - P273 - P280 - P302 + P352 - P308 + P313
• Classi di pericolo: Aquatic Chronic 2 - Carc. 1B - Skin Sens. 1 - STOT RE 2 Inhalation
• Organi bersaglio: Respiratory Tract
• Codice della classe di stoccaggio: 6.1C - Combustible acute toxic Cat.3 / toxic compounds or compounds which causing chronic effects