Tecnai spirit transmission electron microscope

For immuno-EM, 1 × 10⁶ NK cells at different stages form WT mice were isolated. and fixed with 4% paraformaldehyde and 0.05% glutaraldehyde for 2 h on ice and then cryo-sectioned by Tokuyasu method and mounted in copper grids followed by blocking in 1% BSA. Sections were stained with anti-LC3 (30 ng μl⁻¹) antibody (PM036, MBL) and further stained with 10 nM gold linked anti-rabbit IgG. Then the sections were fixed with 2.5% glutaraldehyde and stained with neutral uranyl acetate before coating with 2% methyl cellulose. Images were taken with a FEI Tecnai spirit transmission electron microscope.

Transmission electron microscopy (TEM) was performed at the Center of Electron Microscopy of São Paulo State University (UNESP), Botucatu Biosciences Institute (São Paulo state, Brazil), regarding its previously reported specifications [18 (link)].
Two strains, numbered 21 (C. gattii) and 41 (C. neoformans), were treated with the respective liriodenine MICs in a 24-well plate. The final volumes of liriodenine and inoculum were adjusted to 1 mL, and Karnovsky’s fixative was added after 48-h incubation at 37 °C. Following this period, the material was removed from the fixative and washed three times for 5 min each in 0.1 M phosphate buffer, pH 7.3. The material was immersed in 0.1 M osmium tetroxide, pH 7.3, for 2 h. Next, the material was washed three times for 10 min each in distilled water and immersed in 0.5% uranyl acetate for approximately 2 h. After dehydration in an increasing acetone series, a mixture of Araldite® resin + 100% acetone (1:1) was added, and the material was left to stand at room temperature for 12 h. Pure resin was added for approximately one hour at 37ºC, and the material was embedded. Ultrathin (90 nm) sections were cut from the blocks and counterstained with uranyl acetate in 50% alcohol for 20 min, followed by counterstaining with lead citrate for 10 min. The sections were observed with a Tecnai Spirit transmission electron microscope (FEI Company).

For electron microscopy studies, left ventricle cardiac tissue was fixed in cold 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M sodium cacodylate buffer, treated with 2% osmium tetroxide in 0.1 M sodium cacodylate, en-bloc stained in 3% aqueous uranyl acetate, dehydrated in ethanol, and embedded in epoxy-araldite resin42 (link). 70 nm sections were cut with a Leuica UC6 ultramicrotome and examined with an FEI Tecnai Spirit transmission electron microscope. Mitochondrial size was analysed using ImageJ.