Tecnai 12 electron microscope

For immunogold electron microscopy, M. marinum strains were inoculated from an exponentially growing culture to 7H9 medium with or without detergent at 0.35 OD₆₀₀/ml and incubated without agitation. After overnight incubation, 5 OD-units of bacteria were collected by centrifugation and fixed for 24 hours in 0.2M PHEM (final concentrations: 120mM PIPES, 50mM HEPES, 4mM MgCl₂ 20mM EGTA) buffer with 4% paraformaldehyde and 0.4% glutaraldehyde. M. tuberculosis was grown until mid-logarithmic phase in the presence of 0.05% Tween-80. At this point, samples were collected and fixed as described above, while cells were also re-inoculated in a medium lacking Tween-80 and incubated for 6 days after which samples were collected and fixed. Fixed bacteria were incubated on carbon coated formvar grids for 5 minutes. Grids were stained with different antibodies: anti-EspE polyclonal rabbit serum (produced as described in [67 (link)], quality control in S3A Fig); anti-PIM₆ (F183-24) [6 (link),33 (link)]; anti-ManLAM (55.92.1A1) [6 (link),11 (link)]. Gold-labelled secondary antibodies (Utrecht University) were used and surface labelling was visualized on a Tecnai 12 electron microscope (FEI, Eindhoven, the Netherlands). Plunge freezing and Cryo-EM was performed as described in Sani et al. 2010 [6 (link)].

Samples were processed essentially as described earlier (Peters et al., 2006 (link)). In brief, yeast cells expressing Grh1-2xGFP cultured in starvation conditions (see above) were fixed with 4% paraformaldehyde in PBS for 2 hr. The cells were then washed with PBS/0.02 M glycine, and resuspended in 12% gelatin in PBS, and then embedded in the same solution. The embedded cells were cut in 1 mm blocks and infiltrated with 2.3 M sucrose at 4°C, mounted on aluminum pins, and frozen in liquid nitrogen. The samples were then sectioned and the ultrathin cryosections were picked up in a mixture of 50% sucrose and 50% methylcellulose and incubated with antibodies to Acb1 and GFP (to monitor Grh1) followed by protein A gold (15 nm and 10 nm respectively) in this sequential order. The labelled sections were then imaged in FEI Tecnai-12 electron microscope.

U2OS cells were plated on glass gridded coverslips (Electron Microscopy Sciences, Hatfield, PA) and transfected with indicated plasmids. 24hrs after transfection, cells were fixed in 2% glutaraldehyde, 2 mM CaCl2 in 0.08 M sodium cacodylate buffer, pH 7.2 at RT for 10 minutes and imaged on Zeiss Airyscan to collect light microscopy images. Cells were kept in fixative at 4^oC for 16hrs and postfixed in 2% osmium tetroxide-1.25% potassium ferrocyanide in cacodylate buffer for 30 min followed by 2% osmium in cacodylate buffer for another 30 min and processed for Epon embedding. Cells imaged by Airyscan were localized on the grid (imprinted in the Epon block). Ultrathin sections (60 nm) from the imaged cells were cut and post-stained with uranyl acetate/lead citrate and imaged in a Tecnai 12 electron microscope (FEI, Hillsboro, OR) operating at 80kV equipped with an Ultrascan 4000 digital camera (Gatan Inc, CA).

Larval brains were fixed in 4% paraformaldehyde and 2% glutaraldehyde and embedded as previously described (Orso et al., 2009 (link)). Electron microscopy images were acquired from thin sections under a FEI Tecnai-12 electron microscope at the DeBio imaging Electron Microscopy Facility (University of Padova).

EVs were purified on a density gradient as described above. The EV containing fractions with a density between 1.21–1.07 g/mL were pooled (total volume of 580 µL) and washed with PBS on a 0.5 mL 10 kDa centrifugal filter (Amicon, Merck KGaA, Darmstadt, Germany) as described (Kuipers et al., 2022 (link)). The sample (3 µL) was subsequently applied on a 300 mesh EM grid (Quantifoil R2/2, Jena, Germany) that was previously glow-discharged (2 min in 0.2 mbar air using a EMITECH K950X with glow discharger unit) and vitrified using an EMGP (Leica, Wetzlar, Germany) at room temperature and 100% humidity. Excess sample was removed by blotting once for 1 s with filter paper (Whatman #1). The blotted grid was plunged into liquid ethane (−183°C). After vitrification, the grid was stored under liquid nitrogen until further use. The grid was mounted in a Gatan 626 cryo-holder for cryo-EM imaging. Cryo-EM imaging was performed on a Tecnai 12 electron microscope (FEI Company, Eindhoven, Netherlands) operated at 120 kV. Images were recorded on a 4k×4k Eagle camera (FEI Company) at ×18,000 magnification (pixel size 1.2 nm) between 5 and 10 µm under focus.

Liver and muscle specimens were fixed in a mixture of 2% paraformaldehyde and 1% glutaraldehyde prepared in 0.2 M HEPES. Samples were then post‐fixed in a mixture of osmium tetroxide and potassium ferrocyanide, dehydrated in ethanol and propylene oxide and embedded in epoxy resin as described previously (Polishchuk et al, 2014). 65‐nm‐thin sections were cut using a Leica EM UC7 ultramicrotome. EM images were acquired using a FEI Tecnai‐12 electron microscope (FEI, Eindhoven, the Netherlands) equipped with a VELETTA CCD digital camera (Soft Imaging Systems GmbH, Munster, Germany). Number of mitochondria was counted using the same magnification within 100‐μm square field of view. For each experiment, between 244 and 365 individual mitochondrial diameters were measured from two mice per group.