Jem 1400 electron

Ookinetes were produced as outlined above and purified from uninfected erythrocytes by gradient purification centrifugation on a 63% Nycodenz (in PBS) cushion. In order to more easily visualize the mitochondrial network, we adapted a 3,3′-diaminobenzidine (DAB) staining procedure [43 (link),44 (link)]. Cells were fixed in 4% glutaraldehyde (0.1 M phosphate buffer pH 7.2) for approximately 1 h at 4°C. Fixed cells were then washed extensively (twice washed with 0.1 M phosphate buffer pH 7.2, followed by twice washed with 0.1 M Tris buffer pH 7.5) and resuspended in a solution containing 2 mg DAB and 0.06% (v/v) H₂O₂ in 0.1 M Tris buffer pH 7.5. The reaction was allowed to proceed at room temperature (in the dark) for approximately 80 min and was then washed once with an equivalent volume of distilled water. Prepared cells were postfixed with 1.5% KMnO₄ for 20 min at room temperature, rinsed in water and stained overnight in 0.5% uranyl acetate at 4°C. In preparation for imaging, samples were serially dehydrated with ethanol and embedded in Epon (60°C for 8 h) followed by sectioning. Images were acquired on a JEOL JEM-1400 electron microscope at 80 kV using the TemCam F416 (Tietz Video and Image Processing Systems GmBH, Gautig).

Two µl of the virus solution was placed on a glow-discharged copper grid covered with a continuous carbon film. After 1 min of adsorption, the grid was washed with pure water for several seconds and stained with 3 µl of 2% (w/v) uranyl acetate solution for 1 min. The staining solution was blotted away with Whatman No. 1 filter paper. The grid was air-dried completely before it was examined in a JEOL JEM-1400 electron microscope operating at 120 keV. The micrographs were recorded at various magnifications using a 4K × 4K CMOS camera (TVIPS F-416).