Uranyl oxalate

TEx and TMv fractions were fixed in 2% paraformaldehyde (Serva) and allowed to adsorb onto formvar carbon-coated grids for 20 min. The grids were then washed in PBS (IIET), fixed in 1% glutaraldehyde (Sigma-Aldrich) for 5 min and washed with water (7 × 2 min). Then each grid was transferred to a drop of uranyl-oxalate (4% uranyl acetate and 0.15 M oxalic acid in 1:1 v:v ratio; Sigma-Aldrich) at pH 7 for 5 min. At this stage, samples were counterstained using two protocols: 1. with uranyl acetate or 2. with methylcellulose. 1: The grid was transferred to a drop of 2% uranyl acetate (Chemapol) for 5 min and washed with a drop of water 3 times. Then the grids were allowed to air dry for 10 min. 2: The grids were then embedded in 2% methylcellulose (Sigma-Aldrich) with uranyl acetate (9:1 v:v ratio) for 10 min on ice. The excess of methylcellulose was removed from grids by filter paper and grids were allowed to air dry for 20 min. Preparations were visualized using a JEOL JEM-1200 EX 80 kV TEM.

Methods were modified from Théry et al. (2006) [42 (link)]. 10uL of pooled saliva from uninfected (mock) and DENV2 infected mosquitoes was directly absorbed onto formvar copper grids (Electron Microscopy Sciences) for 10 min and excess saliva blotted away. Next, grids were fixed by floating on a mixture of 2% glutaraldehyde (Electron Microscopy Sciences) and 2% paraformaldehyde (Electron Microscopy Sciences) for 10 min and excess fixative blotted away. Grids were then floated on a solution of uranyl acetate (Electron Microscopy Services) and 0.15M oxalic acid (Sigma-Aldrich) at a 1:1 ratio for 5 min and excess uranyl oxalate blotted away. Lastly, grids were embedded by floating on a solution containing 2% methyl cellulose (Sigma-Aldrich, 25 centripose, M-6385) and 4% uranyl acetate (Electron Microscopy Services) at a ratio of 9:1 for 10 min on ice. Grids were examined in a JEOL JEM-1400 (USA) transmission electron microscope at 80 kV. Images were acquired on a bottom-mounted CCD camera Orius SC2001 (Gatan, Pleasanton, CA).