Morada ccd

RD cell monolayers in 6-well plates (10⁶ cells/well) were harvested after infection with 1MOI EV71 at 0 hpi, 12 hpi, 24 hpi and 48 hpi, and were washed with 1×PBS. The cell pellets were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1.5 h at 4°C, post-fixed in cacodylate buffer containing 1% OsO₄ for 1 h at 4°C. The samples were then stained with 1% uranyl acetate buffer overnight at 4°C in the process of 80% ethanol dehydration during dewatered with gradient ethanol. The cells were embedded in ethoxyline resin (Embed812EMS) and sectioned at a thickness of 60 nm. The sections collected on formver/carbon-coated grids were stained with 2% uranyl acetate and lead citrate buffer for 30 min and observed under an FEI Tecnai G2 Spirit Biotwin TEM at 200 kV. Pictures were captured by Morada CCD and iTEM software (Olympus Optical Co.Ltd., Tokyo, Japan). The brain and skeletal muscle of mice without or with EV71 infection at 7 dpi were sliced into 1 mm³ cube with a sharp blade. The samples were handled as described above.
The rate of mitochondrial dysfunction (%) was calculated by the number of damaged mitochondria in the total number of mitochondria under three different versions.

The infected RD cells were fixed in 2.5% gluteraldehyde in 0.1 M sodium cacodylate buffer (pH = 7.4) for 1 h. The cells were rinsed in sodium cacodylate and those in petri dishes were scraped and spun down in 2% agar. All samples were fixed in 1% osmium tetroxide for 1 h, stained en masse in 2% uranyl acetate in maleate buffer (pH = 5.2) for a further hour, rinsed and dehydrated in an ethanol series, and infiltrated with resin (Embed812 EMS) and baked over night at 60 uC. Hardened blocks were cut using a Leica Ultra Cut UCT. 60 nm sections were collected on formver/carbon-coated grids and contrast stained using 2% uranyl acetate and lead citrate. Samples were viewed on an FEI Tecnai G2 Spirit Biotwin TEM at 200 kV. Images were taken using Morada CCD and iTEM software (Olympus Optical Co. Ltd., Tokyo, Japan).

Sample preparation, immune labeling and image processing for immunoelectron microscopic analysis of B. microti LabS1–infected mouse (CB.17-SCID) red blood cells (mRBCs) were performed as previously described by Thekkiniath et al. (Thekkiniath et al., 2019 (link)). Briefly, B. microti-infected mRBCs were fixed in 4% PFA and frozen using a Leica HMP100 at 2,000 psi. The frozen samples were then freeze-substituted using a Leica Freeze AFS unit starting at −95°C using 1% osmium tetroxide, 1% glutaraldehyde, and 1% water in acetone for 10h, warmed to −20°C for 12h and then to 4°C for 2h. The samples were rinsed in 100% acetone and infiltrated with Durcupan resin (Electron Microscopy Science) and baked at 60°C for 24h. Hardened blocks were cut using a Leica UltraCut UC7, and 60-nm sections were collected on formvar/carbon–coated nickel grids. Resin sections were incubated with anti-BmGPI12 monoclonal antibodies at 1: 100 dilution (overnight), rinsed in buffer, and then incubated with the secondary antibody 10 nm protein A gold (UtrechtUMC) for 30 min. The grids were rinsed and fixed using 1% glutaraldehyde for 5 min, rinsed well in distilled water, and contrast-stained using 2% uranyl acetate and lead citrate. The grids were viewed in an FEI Tencai Biotwin TEM at 80 kV. Images were taken using Morada CCD and iTEM (Olympus) software.