By Andréa C. LeBlanc
McGill Univ., Montreal, Canada. deals state of the art protocols to enquire apoptosis in neurons, how to notice key proteins interested by neuronal apoptosis, recommendations to review synaptosis and observe proapoptotic proteins in situ, and notes at the pitfalls and merits of every technique.
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Extra info for Apoptosis Techniques and Protocols
14. Rinse with PBS (no Tween-20). We recommend using chambered cover glasses, which make immunostaining very easy because of the wells. At this point in the protocol, leave the chambers on the cover glasses and simply cover the cells with PBS or mounting medium; trying to remove the wells from a chambered cover glass is unnecessary, and they break easily. If the high cost of these chambers is prohibitive, then treated cover slips cultured in 24-well plates will work just as well, but will require mounting before microscopy.
Generally, one should see two major bands with molecular weights of 50 and 25 kDa, corresponding to the heavy and light chains of the antibody. Pool the peak antibody fractions. 2. COUPLING OF BAX ANTIBODIES TO SEPHAROSE BEADS 1. 4, at 4ºC. Change the dialyzing solution every 6–8 h for a total of 4 times. Remove the precipitate by centrifugation at 8000 rpm in a Sorvall SA 600 rotor. Determine the A280 of the sample. 3 equals 1 mg/ mL for antibodies. 2. Weigh out appropriate amount of CNBr-activated Sepharose 4 B beads (Sigma).
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.