Kit de detección de la apoptosis in situ con peroxidasa ApopTag

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).

El kit de detección de la apoptosis in situ con peroxidasa ApopTag detecta células apoptóticas in situ al marcar y detectar roturas en la hebra de ADN mediante el método TUNEL. Este kit proporciona suficientes reactivos para realizar la técnica de inmunoperoxidasa de 40 muestras. Los resultados se visualizan utilizando el microscopio de campo claro.

Guardar el kit entre -15°C y -25°C hasta el primer uso. Después de la primera utilización, si el kit va a ser utilizado en los siguientes tres meses, guardar la enzima TdT (90418) entre -15°C y -25°C y el resto de los componentes entre 2°C y 8°C.

Precauciones

1.Los siguientes componentes del kit contienen cacodilato (ácido dimetilarsínico) de potasio como tampón: Tampón equilibrador (90416), tampón de reacción (90417) y enzima TdT (90418). Estos componentes son nocivos en caso de ingestión; evítese el contacto con los ojos y la piel (utilización de guantes y gafas) y lávense de inmediato las zonas de contacto.

2. Los conjugados de anticuerpos (90420) y las disoluciones de bloqueo (nº 10 y nº 13) contienen acida sódica al 0,08 % como conservante.

3. La enzima TdT (90418) contiene glicerol y no se congelará a -20°C. Para una máxima vida útil, no caliente este reactivo a temperatura ambiente antes de su dispensación.

Tampón equilibrador: 3,0 ml de -15°C a -25°C

Tampón de reacción 2,0 ml de -15°C a -25°C

Enzima TdT 0,64 ml de -15°C a -25°C

Tampón de parada/lavado 20 ml de -15°C a -25°C

Anti-digoxigenina-peroxidasa* 3,0 ml de 2°C a 8°C

Cubreobjetos de plástico 100 cada uno. Temperatura ambiente

Nota: Es necesaria la adquisición por separado de DAB (sustrato de la peroxidasa). No se suministra con este kit.

Número de muestras por kit: Se suministra material suficiente para teñir 40 muestras de tejido de aproximadamente 50 mm² cada una cuando se utiliza según las instrucciones. El tampón de reacción se consumirá completamente antes que los demás reactivos cuando los kit se utilizan para muestras montadas en portaobjetos.

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