Spurr s low viscosity resin

For transmission electron microscopy, small fragments of the left kidneys were fixed by immersion fixation in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 h. Afterwards, kidneys were treated with 1% OsO₄ in 0.1 M cacodylate buffer for 1.5 h, dehydrated with graded ethanol solutions, and embedded in Spurr's low-viscosity resin (Electron Microscopy Sciences, Hatfield, PA, USA). Semithin sections were stained with toluidine blue stain. Tissues were sectioned using an Ultracut UCT Microtome (Leica Microsystems) equipped with a diamond knife, mounted on copper grids coated with a formvar film, and stained with uranyl acetate and lead citrate. Ultrathin sections were analyzed on a transmission electron microscope (Hitachi H-7100 Electron Microscope; Hitachi, Tokyo, Japan).
For scanning electron microscopy, kidney samples were fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 h. After treatment with 1% OsO₄ in 0.1 M cacodylate buffer for 1.5 h, the samples were dehydrated with graded ethanol solutions and immersed in isoamyl acetate solution for 30 min. The kidney samples were dehydrated by critical-point drying with liquefied carbon dioxide. Dried samples were sputter-coated with platinum and examined with a scanning electron microscope (Hitachi S-3400 N electron microscope; Hitachi).

Transmission electron microscopy was performed using the Electron Microscopy Facility of Harvard Medical School. A pellet of cells was fixed for at least 2 h at room temperature in 2.5% glutaraldehyde, 1.25% paraformaldehyde and 0.03% picric acid in 0.1 M sodium cacodylate buffer (pH 7.4), washed in 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide/1.5% potassium ferrocyanide for 1 h, washed twice in water, once in maleate buffer and incubated in 1% uranyl acetate in maleate buffer for 1 h, followed by two washes in water and subsequent dehydration in grades of alcohol (10 min each: 50%, 70%, 90%; 2 × 10 min: 100%). The samples were then incubated with propylene oxide for 1 h and infiltrated overnight in a 1:1 mixture of propylene oxide and Spurr’s low-viscosity resin (Electron Microscopy Sciences). The following day the samples were embedded in Spurr’s resin and polymerized at 60 °C for 48 h. Ultrathin sections (about 60 nm) were cut on a Reichert Ultracut-S microtome, transferred to copper grids stained with lead citrate and examined in a JEOL 1200EX transmission electron microscope, and images were recorded with an AMT 2k CCD camera.