Uc6 ultramicrotome

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).

Protoplasts from the calli were generated as previously described [69 ], then protoplast suspensions were dropped onto microscope slides to observe the plastid modes. Light microscopy of various orange tissues of Hongkong kumquats was performed using a frozen sectioning technique with a Leica CM1900 (Leica, Germany). An optical microscope (BX61, Olympus) equipped with a DP70 camera was used in tandem with a differential interference contrast (DIC) technique.
Transmission electron microscopy (TEM) analysis was performed according to Cao et al. [28 (link)]. Samples were prepared using a normal fixation process with 2.5% glutaraldehyde adjusted to pH 7.4, and a 0.1 M phosphate buffer with 2% OsO₄. The preparations were dehydrated and embedded in epoxy resin and SPI-812, respectively. Ultrathin sections obtained with a Leica UC6 ultramicrotome were stained with uranyl acetate and subsequently with lead citrate. Image recording was performed with a HITACHI H-7650 transmission electron microscope at 80 KV and a Gatan 832 CCD camera.
Starch granule morphology was examined with a scanning electron microscope (SEM). The samples were mounted on studs, sputter coated with gold (Balzers, JFC-1600), and examined under a JSM-6390LV SEM (JEOL, Japan).

Petals were fixed in 2.5% (v/v) glutaraldehyde and embedded in LR White resin (London Resin Company, Reading, Berkshire, UK) as described [63] (link). The material was sectioned with a diamond knife using a Leica UC6 ultramicrotome (Leica, Milton Keynes). Semi-thin sections of approx. 500 nm were stained with Toluidine blue for light microscopy, whereas ultrathin sections of approx. 90 nm were collected for electron microscopy. These were picked up on copper grids which had been pyroxylin and carbon-coated. The sections were stained with 2% (w/v) uranyl acetate and 2% lead citrate and viewed in a FEI Tecnai 20 transmission electron microscope (FEI UK Ltd, Cambridge, UK) at 200 kV. TIF digital image files were recorded using an AMT XR60 digital camera (Deben, Bury St Edmunds, UK).
Petals were cryo-fixed and freeze-fractured as described [64] (link), using an ALTO 2500 cryo-system (Gatan, Oxford, England) attached to a Zeiss Supra 55 VP FEG scanning electron microscope (Zeiss SMT, Germany). The sample was imaged at 3 kV and digital TIF files were stored.

The placental tissues were perfused with 0.1 M sodium cacodylate buffer (Electron Microscopy Services, EMS, Hatfield, PA, USA), at pH 7.4. and subsequently fixed with 2% formaldehyde (Merck, Darmstadt, Germany) and 2% glutardialdehyde (Merck, Darmstadt, Germany). Tissue specimens of 1 mm³ were isolated and rinsed over night at 4 °C in perfusion buffer and post fixed in 2% osmium tetroxide in the same buffer. They were dehydrated in a series of graded ethanol (Merck, Darmstadt, Germany), placed into propylene oxide (Sigma-Aldrich, Steinheim, Germany) as an intermedium and embedded in TAAB embedding resin (TAAB, Aldermaston, UK). Leica UC 6 ultramicrotome (Leica, Vienna, Austria) was used to obtain 70 nm thick tissue sections. Tissue sections were visualized with a Zeiss EM 900 transmission electron microscope (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany) at an acceleration voltage of 80 kV. Scale bars and structure indicating arrows were consecutively inserted to the images with ImageJ 1.50e [81 (link)].

Malassezia sympodialis yeasts were cultured as described above on mDixon agar plates, scraped off using a loop, and suspended in a minimal amount of distilled water to form a paste. Approximately 2 µl of each sample was processed by HPF using a Leica EM PACT 2 (Leica, Milton Keynes, UK). After HPF, samples were transferred to a Leica AFS 2 embedding system for freeze substitution in a solution with 2% OsO₄ in 100% acetone for 40 min. Samples were then placed in 10% Spurr resin:acetone (TAAB Laboratories, Berks, UK) for 72 h followed by 30% Spurr resin overnight, 8 h of 50% Spurr resin, 12 h of 70% Spurr resin, 90% Spurr resin for 8 h, and finally embedded in 100% Spurr resin at 60°C for at least 24 h. Sections (90 µm) were cut with a diamond knife onto nickel grids using a Leica UC6 ultramicrotome. Sections were contrast stained with UranyLess EM stain (TABB Group, New York, US) and lead citrate in a Leica AC20 automatic contrasting instrument. Samples were imaged using a JEM 1400 plus transmission electron microscope with AMT ultraVUE camera (JEOL, Welwyn Garden City, UK). These experiments were performed on three biological replicates.

Renal cortex was processed as previously described.⁶¹ Sections were cut at 60nm on a Leica UC6 ultramicrotome (Leica) and were imaged at 80 kV on a Jeol JSM 1011 transmission electron microscope (Jeol) equipped with an Olympus Morada (Olympus, Japan) digital camera. Quantification of foot process width and GBM expansion was assessed as previously described.⁶²

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.