By Andréa C. LeBlanc
During this revised and extended moment variation, professional specialists describe in step by step aspect their most sensible cutting-edge concepts for learning neuronal phone loss of life. those conveniently reproducible tools clear up a large choice of analysis difficulties, together with the detection of the major proteins focused on neuronal apoptosis (Bax protein, cytochrome c, and caspases), the direct evaluation of the function of pro-apoptotic proteins in neurons by means of viral infections and microinjections, and the detection of pro-apoptotic proteins in situ. There also are hands-on tools for the learn of apoptosis mechanisms in neuronal booths, for learning synaptosis, and for developing gene expression profiles in neurodegenerative mind tissues through the use of DNA microarrays.
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Additional info for Apoptosis: Techniques and Protocols (Neuromethods)
These fractions can also be probed for many proteins that are cleaved in apoptosis. In addition, the cytoplasmic extracts can be assayed directly for caspase activity using fluorescent substrates. 2. Disadvantages Consistency and attention to detail make this process technically demanding. 3. 4. 5. Homogenization Buffer 1. 2. 3. 4. 6 g of sucrose in 50 mL dH2O. Dissolve 2 tablets of complete protease inhibitor cocktail. 4). Add 1 mL of 1 M KCl. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Cytochrome-c Release 5.
Disadvantages 1. Requires access to a confocal microscope or a good fluorescence microscope. 2. Fluorescence will fade. 3. Reagents and Solutions See Table 1. 4. Antibodies for Immunostaining See Table 2. 5. Paraformaldehyde Fixative 1. 0 g paraformaldehyde in ~60 mL H 2O with one drop of 10 N NaOH. 2. Warm to no more than 60ºC, on a heating stir plate. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 24 Ethell and Green Table 1 Reagents and Solutions for Immunostaining Reagent Catalog number Vendor Bovine serum albumin (BSA) Chambered cover glass Cover slips CytosealXYL BP1605-100 155409 12-545-82 8312-4 DAPI Dimethyl sulfoxide (DMSO) Normal goat serum (NGS) Paraformaldehyde 10X phosphate-buffered saline (PBS) Tween-20 Vectashield mounting medium D8417 D2650 G9023 T353-500 70011-044 Vectashield + DAPI H-1200 Fisher Nalge-NUNC Fisher Stephens Scientific (Kalamazoo, MI) Sigma Sigma Sigma (St.
Natl. Acad. Sci. USA 94, 3668–3672. 19. -T. and Youle, R. Y. (1998) Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J. Biol. Chem. 273, 10,777–10,783. 20. , Wei, M. , and Korsmeyer, S. J. (1998) Enforced dimerization of Bax results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 17, 3878–3885. 21. Wolter, K. , Smith, C. , and Youle, R. J. (1997) Movement of Bax from the cytosol to mitochondria during apoptosis.
Apoptosis: Techniques and Protocols (Neuromethods) by Andréa C. LeBlanc