Scoring of Apoptotic Nuclei


 * Hermaphrodites were picked as late-stage L4s and allowed to age for 22-26 hours.
 * They were mounted in M9 on 1.5% agarose pads containing 0.2 mM levamisole and their gonads examined by fluorescence microscopy for cells completely surrounded by ced1::GFP. 30-40 gonads are scored per genotype
 * make agar pads that contain levamisole (the stock solution is 1000X; this immobilizes the worms) fresh every-time; for dumpy worms this stock is 2000X
 * keep the agar at ~95 degrees while making each pad
 * flank the slide that you are making your pad on with a slide and coverslip on either side
 * put a drop of 1.5% agarose + levamisole on a slide and drop a 22X50 coverslip perpendicular to the slide on top to flatten.
 * leave for 3 minutes
 * slide the coverslip off and add drop of oil
 * place worms on the pad quickly so it doesn’t dry out
 * put 22X22 coverslip on top
 * seal with agarose
 * add 10-25 ul M9 underneath coverslip
 * visualize on Deltavision with 100X objective