Ultracut uc6

Tissue was fixed in 4% paraformaldehyde (w/v) and 2% glutaraldehyde (w/v; phosphate buffered saline) overnight. After fixation tissue was cut into 100 μm thin slices, thoroughly washed in phosphate buffer (0.1M) and incubated with 1% osmium tetroxide for 60 minutes. After washing in graded ethanol (up to 50% /v/v)) for 10 minutes each, slices were incubated with uranyl acetate (1% (w/v) in 70% (v/v) ethanol) overnight. Slices were then dehydrated in graded ethanol (80%, 90%, 96% for 10 minutes each, and 2x 100% for 10 minutes). After two washing steps in propylene oxide for 5 minutes each, the slices were incubated with durcupan/propylenoxid (1:1 for 1 hour) following durcupan only overnight at room temperature. Ultrathin sections of 20–40 nm were prepared at a Leica UC6 Ultracut. Sections were mounted onto copper grids (Plano) and contrasted using Pb-citrate for 3 minutes. Transmission electron microscopy was performed at a Zeiss Leo 906 equipped at 6000x magnification. In total, cortical access tissue from 7 individuals who underwent clinically indicated neurosurgical procedures, e.g., for tumors or epilepsy, were processed and 1 to 6 acquisitions were obtained from each patient. Each acquisition included at least one labeled dendritic segment and synaptic contact. Up to 40 images of the same dendritic spines on consecutive sections were acquired and saved as TIF-files.

hiPSC-ROs were fixed in 4% paraformaldehyde, 2,5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7,2–7,4) for 1,5 h and washed with the same buffer. Then, the samples were post-fixed with 2% osmium, rinsed, dehydrated and embedded in Durcupan resin (Fluka, Sigma-Aldrich, St. Louis, USA). Serial semi-thin sections (1.5 µm) were cut with an ultramicrotome Ultracut UC-6 (Leica, Heidelberg, Germany), mounted onto slides and stained with 1% toluidine blue in 1% Borax and analyzed under a light microscope. Ultrathin sections (0.07–0.09 µm) were prepared with the Ultracut and stained with lead citrate. Finally, photomicrographs were obtained under a transmission electron microscope FEI Tecnai G2 Spirit (FEI Europe, Eindhoven, Netherlands) using a digital camera Morada (Olympus Soft Image Solutions GmbH, Münster, Germany). Sample processing was performed by CIPF Electron Microscopy Core Facility.

Cell samples were fixed in 4% paraformaldehyde plus 1% glutaraldehyde in 0.1 M phosphate buffer overnight. After contrasting using 1% osmium tetroxide in 0.1 M phosphate buffer (45 minutes at room temperature) and 1% uranyl acetate (in 70% ethanol, room temperature), samples were dehydrated in an ascending ethanol series and embedded in epoxy resin (Durcupan, Sigma-Aldrich). Ultrathin sections of approximately 70 nm thickness were prepared using a Leica Ultracut UC6. For imaging, a Philips CM100 transmission electron microscope was used.

MEFS cells were seeded at 3×10⁴ cells per chamber on Lab-Tek chamber slides of 4 wells (Nalge Nunc International). Then, cells were fixed for 1 h in 3.5% glutaraldehyde at 37°C and postfixed for 1 h in 2% OsO4 at room temperature. Cellular staining was performed at 4°C for 2 h in 2% uranyl acetate in the dark. Finally, cells were rinsed in sodium phosphate buffer (0.1 M, pH 7.2), dehydrated in ethanol and infiltrated overnight in Araldite (Durcupan, Fluka, Buchs SG, Switzerland). Following polymerisation, embedded cultures were detached from the chamber slide and glued to Araldite blocks. Serial semi-thin (1.5 µm) sections were cut with an Ultracut UC-6 (Leica, Leica, Heidelberg, Germany), mounted onto slides and stained with 1% toluidine blue. The selected semi-thin sections were glued (Super Glue, Loctite) to araldite blocks and detached from the glass slide by repeated freezing (in liquid nitrogen) and thawing. Ultrathin (0.07 µm) sections were prepared with the Ultracut UC-6 and stained with lead citrate. Finally, photomicrographs were obtained under a transmission electron microscope (FEI Tecnai Spirit G2) using a digital camera (Morada, Soft Imaging System, Olympus). Then, mitochondrial width was quantified by using the Image J Java-based image processing software (NIH).

RPE-1 cells grown on 35 mm glass bottom dishes (MatTek Corporation) were co-transfected with a plasmid expressing GFP-Centrin2 and indicated siRNAs for 48 h. The culture media was changed to Opti-MEM reduced serum media (Life Technologies) to promote the formation of primary cilia for 24 h. We then performed correlative light and electron microscopy strategy to observe the structure of TZ. Briefly, we first selected centrosomes by light microscope and then the same centrosome was imaged on the electron microscope. Cells were fixed for 1 h at room temperature with 4% paraformaldehyde and 2% glutaraldehyde buffered in PBS buffer. Samples were imaged with ×63/1.40 oil objective on Zeiss LSM 880 system. After target cells were selected and marked on the light microscope, cells were incubated in 0.15% Tannic acid in PBS buffer for 1 min, post-fixed in 2% OsO₄ in sodium cacodylate buffer for 1 h on ice, prestained with 1% uranyl acetate diluted in ddH₂O for 1 h at 4 °C, dehydrated in a graded series of ethanol, infiltrated with EPON812 resin (Electron Microscopy Sciences), and then embedded in the resin. Serial sections (60–80 nm thickness) were cut on a microtome (Ultracut UC6; Leica) and stained with 1% uranyl acetate as well as 1% lead citrate. Samples were examined on a JOEL transmission electron microscope.