Ccd camera

Purified MERS-CoV and SARS-CoV S proteins were adsorbed by flotation for 2 minutes onto a freshly discharged 400-mesh carbon parlodion-coated copper grid (Poly-Sciences, Warrington, PA). The grids were rinsed with buffer containing 20mM Tris, pH 7.4, and 120mM KCl and negatively stained with 1% phosphotungstic acid, then dried by aspiration. Virus-like particles were visualized on a Hitachi H-7600 transmission electron microscope (Hitachi High Technologies America, Schaumburg, IL) operating at 80 kV and digitally captured with a CCD camera at 1K×1K resolution (Advanced Microscopy Techniques Corp., Danvers, MA).

VLP samples were adsorbed onto a freshly discharged 400 mesh carbon parlodion-coated copper grids (Poly-Sciences, Warrington, PA). The grids were rinsed with buffer containing 20 mM Tris, pH 7.4, and 120 mM KCl and negatively stained with 1% phosphotungstic acid, then dried by aspiration. VLPs were visualized on a Hitachi H-7600 transmission electron microscope (Hitachi High Technologies America, Schaumburg, IL) operating at 80 kV and digitally captured with a CCD camera at 1k×1k resolution (Advanced Microscopy Techniques Corp., Danvers, MA).

C albicans cells were processed for ultrastructure studies, with modifications in previously described protocol [28 (link)]. After 24h of respective treatments in YPD broth, C albicans cells were initially fixed for overnight with 500 µl of 3% glutaraldehyde/0.1 M phosphate buffer followed by fixation with same fixative with 0.15% tannic acid for 1h. After three rinses in 0.1 M phosphate buffer, the specimens were subjected to secondary fixation with 1% osmium tetroxide/0.1 M phosphate buffer for 1h. Samples were then rinsed for five times with distilled water followed by Enbloc staining with 1% uranyl acetate in distilled water for 1h. Samples were thoroughly rinsed in distilled water and then dehydrated through a range of 70–100% ethanol. After dehydration, samples were infiltrated with acetone and embedding resin (50:50) for 48 h. Specimens were then embedded with 100% resin (Embed 812, Electron Microscopy Sciences, Hatfield, PA) and polymerized overnight. Sections were cut, 80–85 nm and placed on copper mesh grids and viewed on Transmission Electron Microscope (TEM) (ThermoFisher, Tecnai Spirit, Hillsoboro, OR) and images were captured with CCD camera (Advanced Microscopy Techniques Danvers, MA). Analysis of cell wall thickness was done with TEM images using ImageJ software (n = 20 cells).