Freeze afs unit

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.

Samples fixed in 4% PFA were 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 10 h, warmed to −20°C for 12 h and then to 4°C for 2 h. The samples were rinsed in 100% acetone and infiltrated with Durcupan resin (Electron Microscopy Science) and baked at 60°C for 24 h. Hardened blocks were cut using a Leica UltraCut UC7, and 60-nm sections were collected on formvar/carbon–coated nickel grids.

To examine the ultrastructure of apq12Δ (CPL1326) and apq12Δchm7Δ (CPL1327) strains, unfixed cells were high-pressure frozen using a Leica HMP100 at 2,000 psi and freeze-substituted using a Leica Freeze AFS unit using 1% osmium tetroxide and 1% glutaraldehyde. Samples were infiltrated with durcupan resin (Electron Microscopy Science) and cut in 100 nm thick sections using a Leica UltraCut UC7. Sections were collected on formvar/carbon coated nickel grids and stained with 2% uranyl acetate and lead citrate. Grids were imaged in a FEI Tecnai Biotwin TEM at 80 kV with a Morada CCD camera and iTEM (Olympus) Software.