Glutaraldehyde

Cells were pelleted and fixed for 4 h in 2.5% glutaraldehyde (Euromedex, Mundolsheim, France) in 0.1 M sodium cacodylate buffer, pH 7.2 (Euromedex). Cells were then rinsed with sodium cacodylate buffer and postfixed in 1% osmium tetroxide (Euromedex) for 1 h at RT. Samples were washed and then dehydrated through a graded series of ethanol solutions to water followed by propylene oxide and then infiltrated in 1:1 propylene oxide/poly Bed 812 (Euromedex). Samples were kept overnight embedded in Poly Bed 812, mounted in molds, and left to polymerize in an oven at 56°C for 48 h. Ultrathin Sects. (70–90 nm) were obtained with a Reichert-Jung Ultracut S microtome (Wien, Austria). Sections were stained with uranyl acetate and lead citrate and examined with a CM12 transmission electron microscope (Philips, Eindhoven, The Netherlands).

Cell samples were fixed for 1 h in 2.5% glutaraldehyde (Euromedex, Mundolsheim, France), diluted in 0.1 M cacodylate buffer (Euromedex), pH 7.2, and washed twice in cacodylate buffer. Cells were then postfixed 2 h in 2% osmium tetraoxide, washed with distilled water, and stained with 0.5% uranyl acetate overnight. Samples were then dehydrated in successive ethanol washes, rinsed in propylene oxide, and embedded overnight in 1:1 propylene oxide/Spurr’s resin and left to polymerize at 56 °C for 48 h. Ultrathin sections of 70–90 nm were cut with an ultramicrotome, EM UC7 (Leica), stained with uranyl acetate plus lead citrate, and viewed using a JEM1010 transmission electron microscope (JEOL, Japan).