By Isabelle Martinou, Harald Frankowski, Marc Missotten (auth.), Judes Poirier (eds.)
In Apoptosis options and Protocols major specialists supply investigators in any respect degrees of expertise an fundamental advent to the fundamentals of apoptosis, in addition to crucial information of the tools utilized in its research. those experts conceal such vital subject matters because the histological, organic, and molecular standards for apoptosis and programmed cellphone demise; necrosis and apoptosis within the CNS; and mobile, invertebrate, animal, and human types of apoptosis in Alzheimer's disorder, AIDS, and stroke. The thoughts they describe study the severe steps occupied with the apoptotic technique, and contain PCR research of cell-cycle-regulated proteins, histochemical research of DNA legislation, DNA laddering research, and cytochemical changes of residing cells.
Apoptosis options and Protocols presents a wide selection of vital tools for either experimental and medical research. it's guaranteed to function an illuminating creation to the elemental principles at the back of the phenomena of apoptosis and necrosis, in addition to a key technical reference at the major methodologies utilized in the sphere.
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Extra info for Apoptosis Techniques and Protocols
In most of our work, we have used a modified version of the TUNEL method described by Gavrieli et al. (1992). Slide mounted tissue sections are air dried and then placed in 100% methanol for 15 min. The slides are then placed in a 3% solution of hydrogen peroxide for 15 min. These two steps are critical because they quench endogenous peroxidase activity. This is particularly important in our work with neonatal material, in which tissue is immersion-fixed; many peroxidase-positive red blood cells are presents in this tissue, making it virtually impossible to identify apoptotic cells.
The slides are placed in humid chambers and incubated at 4°C. 4. Wash slides in PBS 3 x 10 min. 5. Incubate in appropriate fluorescent 2” antibody for l-2 h at room temperature. 6. Wash in PBS 3 x 10 min. 7. Coverslip with glycerol phosphate buffer mixture. Acknowledgments This work was supported by an NIH Shannon Award to Thomas Mahalik. -W. and Oppenheim R W. -W and Oppenheim R W. (1978) Cell death of motorneurons m the chick embryo spinal cord II. A quantitative and qualitative analysis of degeneration in the ventral dorsal root, including evidence for axon outgrowth and limb innervation prior to cell death I Comp Neural 177,59-86.
The cells are then washed and placed on slides. Figure 3A shows cresyl violetstained thymocytes illuminated with a rhodamine filter set. Detection of Cell Death Fig. 3. TUNEL-labeling of immature thymocytes in cell cultures. (A) Thymocytes were stained with a dilute solution of cresyl violet that labels all cells. Arrows highlight double-labeled cells. The cells are visualized with the rhodamine filter cube. (B) Fluorescent TUNEL-labeling of cells visualized with fluorescein-tagged avidin. Two apoptotic cells are highlighted by arrows.
Apoptosis Techniques and Protocols by Isabelle Martinou, Harald Frankowski, Marc Missotten (auth.), Judes Poirier (eds.)