Cm10 tem

L4 larvae were transferred to 2.5% glutaraldehyde in 0.1 M Sodium Cacodylate buffer pH 7.2. for 10 min. The tail and head of each worm were dissected out and the main body was transferred to fresh 2.5% glutaraldehyde in 0.1 M Sodium Cacodylate buffer and kept at 4 o C overnight. Samples were processed and analyzed at the University of Massachusetts Medical School Electron Microscopy core facility according to standard procedures. Briefly, the samples were rinsed three times in the same fixation buffer and post-fixed with 1% osmium tetroxide for 1h at room temperature. Samples were then washed three times with ddH2O for 10 minutes and then dehydrated through a graded ethanol series of 20% increments, before two changes in 100% ethanol. Samples were then infiltrated first with two changes of 100% Propylene Oxide and then with a 50%/50%
propylene oxide/SPI-Pon 812 resin mixture. The following day, five changes of fresh 100% SPI-Pon 812 resin were performed before the samples were polymerized at 68 o C in flat pre-filled embedding molds. The samples were then reoriented, and thin sections (approx. 70nm) were placed on copper support grids and contrasted with Lead citrate and Uranyl acetate. Sections were examined using a CM10 TEM with 100Kv accelerating voltage, and images were captured using a Gatan TEM CCD camera.