Jem 1400 lab6 transmission electron microscope

For thin-section TEM examination of embedded RBC vesicles, the samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4). Samples were then washed with the same buffer twice and post-fixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide in the same buffer, dehydrated through a graded ethanol series to 100%, and embedded in Eponate 12 resin (Ted Pella Inc., Redding, CA). Ultrathin sections were cut on a Leica UltraCut S ultramicrotome (Leica Microsystems Inc., Buffalo Grove, IL) at 70 nm, and counter-stained with 4% aqueous uranyl acetate and 2% lead citrate. Sections were examined using a 120 kV JEOL JEM-1400 LaB₆ transmission electron microscope (JEOL, Ltd., Japan) equipped with a Gatan 2k x 2k US1000 CCD camera (Gatan, Inc., Pleasanton, CA).

For TEM examination of endothelial cell cultures, the samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4). Samples were then washed with the same buffer twice and post-fixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide, dehydrated through a graded ethanol series to 100%, and embedded in Eponate 12 resin (Ted Pella Inc., Redding, CA). Ultrathin sections were cut on a Leica UltraCut S ultramicrotome (Leica Microsystems Inc., Buffalo Grove, IL) at 70 nm, and counter-stained with 4% aqueous uranyl acetate and 2% lead citrate. Sections were examined using a 120 kV JEOL JEM-1400 LaB6 transmission electron microscope (JEOL, Ltd., Japan).

The ND were diluted to 0.005 mg/ml based on the MSP1D1 protein concentration. 5 µl of the sample was deposited onto a freshly carbon coated and plasma-treated hexagonal copper EM grid (300 mesh, Ted Pella, Inc., CA, USA) and stained with 1% Uranyl acetate aqueous solution. The rpFVIII-ND complexes were prepared by mixing the initial rpFVIII and ND solutions (at 1 mg/ml concentration each) at 1:1 weight ratio. After 5 min incubation at room temperature, the samples were diluted to a final concentration of 0.002 mg/ml and stained with 1% Uranyl acetate. Digital electron micrographs were collected with a JEM1400-LaB6 transmission electron microscope (JEOL, Ltd) operated at 120 kV with a 2048 × 2048 pixel Ultrascan CCD camera at a final magnification of 84,000×.