Cm100 electron microscope

SNS pellets were prepared from cortical tissue of 1-month-old C57/Bl6 mice as previously mentioned before and submitted to imaging facility at ZMB UZH. Briefly, SNS pellet prepared were resuspended in 2X fixative (5% Glutaraldehyde in 0.2 M Cacodylate buffer) and fixed at RT for 30 mins. Sample was then washed twice with 0.1 M Cacodylate buffer before embedding into 2% Agar Nobile. Post-fixation was performed with 1% Osmium 1 h on ice, washed three times with ddH₂O, dehydrated with 70% ethanol for 20 mins, followed by 80% ethanol for 20 mins, 100% for 30 mins, and finally Propylene for 30 mins. Propylene: Epon Araldite at 1:1 were added overnight followed by addition of Epon Araldite for 1 h at RT. Sample was then embedded via 28 h incubation at 60 °C. The resulting block was then cut into 60 nm ultrathin sections using ultramicrotome. Ribbons of sections were then put onto TEM grid and imaged on TEM - FEI CM100 electron microscope (modify).

Lung capillary EGL was visualized in WT mice by a method previously described by Van den Berg et al. [42 (link)]. For the comparison sham vs. untreated, mice (analysis at 16 and 24 hours after the induction of sepsis) were fully anesthetized and perfused with cardioplegic solution and primary fixative solutions, followed by perfusion with primary fixative solution containing 0.05% Alcian blue. Then, the lung was dissected out and fixed in fixative solution (4% PFA and 1% GA) and post fixed (1% osmium tetroxide and 1% lanthanum nitrate), followed by 1% aqueous uranyl acetate. After being dehydrated in alcohol and propylene oxide, it was embedded in Poly/Bed 812. Ultra-thin sections of capillaries were cut on a Leica EM UC7 ultramicrotome (Polysciences, Warrington, PA, USA), and cross-sections on copper grids were visualized with a Philips (FEI) CM-100 electron microscope at 60kV and quantified with the ImageJ software. The EGL of the lung capillary was semi-quantified by a masked investigator. The total area of the EGL was determined in a single mouse for each measured timepoint, using the ImageJ software, and the numbers were normalized to the perimeter of the capillary (total 500 micrometers of capillaries per timepoint).

TEM was performed with a Philips CM100 electron microscope (FEI Deutschland GmbH, Frankfurt/Main, Germany). For ultrathin sectioning, Dictyostelium cells were fixed and embedded as described previously [18 (link)]. For negative staining, 4-µL aliquots of the dialyzed protein solution were adsorbed for 2 min to pioloform-coated copper grids, fixed for 2 min with 2.5% glutaraldehyde in assembly buffer and stained with 1% uranyl acetate.

Copper grids (carbon coated, 400 mesh (Electron Microscopy Sciences, Hatfield PA)) were glow discharged and inverted on a 7 μl aliquot of sample for 3 minutes. Excess sample was removed and the grids immediately placed briefly on a droplet of double distilled water followed by 1% uranyl formate solution for 2 minutes. Excess stain was removed and the grid allowed to dry thoroughly. Grids were then examined on a Philips CM100 electron microscope (FEI, Hillsbrough OR) at 80 kv and images collected using a Megaview III ccd camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany).

In situ hybridizations and TEM were carried out as described (Franco et al., 2012 (link); Tiveron et al., 1996 (link)). In situ probes are summarized in Table S1. Bright-field images were captured using an Olympus AX70 microscope. Sections for TEM were examined on a Philips CM100 electron microscope (FEI) at 80 kV. Images were collected using a Megaview III CCD camera (Olympus Soft Imaging Solutions).