By Andréa C. LeBlanc
McGill Univ., Montreal, Canada. bargains state of the art protocols to enquire apoptosis in neurons, the right way to become aware of key proteins eager about neuronal apoptosis, strategies to check synaptosis and discover proapoptotic proteins in situ, and notes at the pitfalls and benefits of every technique.
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11. 4 µg/mL Hoechst 33342 for 20 min at room temperature. 12. Rinse the cover slips twice in PBS. 13. , Geltol, Shandon, PA). 14. Assess the extent of cell death by measuring the number of fragmented and shrunken nuclei under a fluorescence microscope. Count 400–500 cells per condition. If caspases are involved, the samples incubated in the presence of the caspase inhibitor should display a significant reduction in the extent of cell death. 15. Apoptosis (and inhibition of apoptosis) can be confirmed using various methods.
11. Stripping Buffer 1. 2. 3. 4. 5. 6. Place 50 mL dH2O into a 100-mL flask, with stir bar. 8). Add 2 g SDS. Add 1 mL of 2-mercaptoethanol. Bring to 100-mL final volume with dH2O. Store at room temperature. 12. Preparation of Cytosolic and Mitochondria Fractions These procedures should be done with ice-cold buffers and kept on ice throughout. 1. Start with 107 neurons/sample and scale down as appropriate. Cultures should be rinsed gently 3X with cold PBS. Neurons cultured on polylysine + laminin can be easily dislodged with a pipetman, which is best as it helps maintain cell integrity.
3. Immunoprecipitation of Bax from Detergent-Solubilized Membranes 1. , Triton X-100, Nonidet P-40, and Chaps) at a concentration of 1–5 × 107 cells/mL. 2. Spin down the unsolubilized material at 12,000 rpm using a Sorval SA 600 rotor. 3. Incubate 1–3 mL cell lysate with 150 µL antibody beads that have been pre-equilibrated in the solubilization buffer. Allow the incubation to proceed for 3 h at 4ºC with constant but gentle rotations. 4. Spin the beads down, remove the supernatant, and wash the beads extensively with 2 washes of 10 mL solubilization buffer.