Empact2 high pressure freezer and rapid transport system

Cells were fixed by high-pressure freezing with an EMPACT2 high-pressure freezer and rapid transport system (Leica Microsystems Ltd.). Cells were freeze-substituted in substitution reagent (1% [wt/vol] OsO₄ in acetone) with a Leica EMAFS2 and embedded in Spurr resin (Sigma). Additional infiltration was provided under a vacuum at 60°C before embedding in Leica FSP specimen containers and polymerized at 60°C for 48 h. Sections (60 nm) were prepared with a Diatome diamond knife on a Leica UC6 ultramicrotome and stained with uranyl acetate and lead citrate for examination with a JEOL 1400 plus transmission microscope (JEOL UK Ltd., Hertfordshire, United Kingdom) and imaging with an AMT UltraVUE camera (Deben).

Yeast and hyphal C. albicans samples were prepared using high-pressure freezing by EMPACT2 high-pressure freezer and rapid transport system (Leica Microsystems Ltd., Milton Keynes, United Kingdom). Cells were freeze-substituted in 1% acetone (w/v) OsO₄ by using a Leica EMAFS2 prior to embedding in Spurr’s resin and polymerizing at 60°C for 48 h. Ultrathin sections were cut using a Diatome diamond knife on a Leica UC6 ultramicrotome and sections were mounted onto formvar coated copper grids. Subsequently, sections on formvar coated copper grids were blocked in blocking buffer (PBS + 1% (w/v) BSA and 0.5% (v/v) Tween20) for 20 min prior to incubation in three washes in binding buffer (150 mM NaCl, 10 mM Tris pH 7.4, 10 mM CaCl₂ in sterile water, 1% FCS) for 5 min. Sections were then incubated with Fc chimeric proteins (5 μg/ml for yeast and 10 μg/ml for hyphae) for 90 min before six washes in binding buffer for 5 min. Fc protein binding was detected by incubation with Protein A conjugated to 10 nm gold (Aurion) (diluted 1:40 in PBS + 0.1% (w/v) BSA) for 60 min prior to six 5 min washes in PBS + 0.1% (w/v) BSA followed by three, 5 min washes in PBS, and three, 5 min washes in water. Sections were stained with uranyl acetate for 1 min prior to three 2 min washes in water and were left to dry. TEM images were taken using a JEM-1400 Plus using an AMT UltraVUE camera.

Candida spp. yeast cells were grown in YPD at 30°C for 6 h and HPF was carried out as described previously (Walker et al., 2010 (link)) with the following modifications. Briefly, samples were prepared by high-pressure freezing with an EMPACT2 high-pressure freezer and rapid transport system (Leica Microsystems Ltd., Milton Keynes, United Kingdom). After freezing, cells were freeze-substituted in substitution reagent (1% [wt/vol] OsO₄ in acetone) with a Leica EMAFS2. Samples were then embedded in Epoxy resin and additional infiltration was provided under a vacuum at 60°C before embedding in Leica FSP specimen containers and polymerizing at 60°C for 48 h. Semithin survey sections, 0.5 μm thick, were stained with 1% toluidine blue to identify areas containing cells. Ultrathin sections (60 nm) were prepared with a Diatome diamond knife on a Leica UC6 ultramicrotome and stained with uranyl acetate and lead citrate for examination with a Philips CM10 transmission microscope (FEI UK Ltd., Cambridge, United Kingdom) and imaging with a Gatan Bioscan 792 (Gatan United Kingdom, Abingdon, United Kingdom). Image J was used to measure the thickness of the inner (chitin and glucan) and outer cell wall by averaging measurements for 30 cells, for each condition.