Ultracut uc7

For electron microscopy, a separate cohort of three males and three females per experimental group (either exposed to saline or Poly I:C at E9.5) was anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and perfused with 3.5% acrolein and 4% PFA (Bisht et al., 2016a (link)) at P80-P90. Fifty-micrometer thick transverse sections from Bregma 2.12–1.64, based on the stereotaxic atlas of Paxinos and Franklin (4th edition), cut with a vibratome, were processed as previously described (Bisht et al., 2016b (link)). Briefly, transverse sections were washed in PBS, quenched, and processed for IBA1 immunostaining. They were blocked and incubated overnight in primary antibody, incubated with goat anti-rabbit secondary antibody conjugated to biotin (#111-065-003, Jackson ImmunoResearch, West Grove, PA, USA) for 1.5 h, and then with ABC Vectastain (1:100, Vector Laboratories, #PK-6100), followed by diaminobenzidine (0.05%) and hydrogen peroxide (0.015%). The sections were post-fixed in 1% osmium tetroxide, dehydrated in ethanol, and embedded with Durcupan resin between ACLAR films (EMS) at 55°C for 72 h. Areas of interest were cut at 65–80 nm using an ultramicrotome (Leica Ultracut UC7). Ultrathin sections were collected on mesh grids and examined at 80 kV with a FEI Tecnai Spirit G2 transmission electron microscope.

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.