Em uc7 microtome

For transmission electron microscopy (TEM) imaging, the PMV pellet was fixed with 2.5% GA (cat. no. G5882, Sigma-Aldrich, St. Louis, USA) in 0.1 M cacodylate buffer (cat. no. C4945, Sigma-Aldrich, St. Louis, USA) for 2 h at RT. Then, samples were postfixed in 1% osmium tetroxide solution (1 h), dehydrated by passing through a graded ethanol series, and embedded in PolyBed 812 at 68 °C. Ultrathin sections were collected on 300-mesh grids or on one slot made from copper; the latter was additionally covered with a formvar film. For cutting, the Leica EM UC7 microtome was used. Then, the sections were contrasted using uranyl acetate and lead citrate. For observation, the JEOL JEM 2100HT electron microscope (Jeol Ltd., Tokyo, Japan) was used at an accelerating voltage of 80 kV.

Small blocks of tissue (0.5×0.5×1 mm) containing secretory cavities at different developmental stages were fixed in a PBS solution (pH 7.2) containing 4% paraformaldehyde and 0.5% glutaraldehyde, and embedded in LR White resin (Sigma). A Leica EM UC7 microtome was used for cutting the sections to a thickness of 70–80 nm.
A nickel grid containing the sections was washed three times with a droplet of PBS-Tween buffer solution for 5 min, and then blocked with 1% BSA for 20 min. The grid was washed three times with PBST for 5 min each and was then floated on PBST containing the anti-CgCaN-specific polyclonal antibodies (primary antibodies, 1:10 v/v) and left to incubate for 3 h at 37 °C. The grid was again washed three times with PBST for 5 min each, then floated on PBST containing colloidal gold antibodies (secondary antibodies, 1:50, v/v; 10-nm gold particles; Sigma-Aldrich) and incubated for 1 h at 37 °C. It was then washed three times with PBST for 5 min each, and twice with d₂H₂O for 5 min each. The grid was then stained with uranyl acetate and lead citrate. Control samples were prepared in a similar manner. In control A, pre-immunization serum was used to replace the primary antibody, and in control B the primary antibody was replaced with PBS. The sections were examined and photographed using a Philips Fei-Tecnai 12 TEM.

Changes in morphology of organelles were observed by TEM analysis. After 8 h of incubation in SD medium containing the required amino acids and uracil with or without ethanol, yeast cells were harvested from 5 ml of cultures by centrifugation at 8000 × g for 3 min and washed twice with 100 mM PBS (pH 7.2). The cells were resuspended in 5 ml of 2.5% (v/v) glutaraldehyde in PBS and pre-fixed overnight at 4 °C. The cell pellets were washed three times with PBS (20 min for each time) and then resuspended in 0.5 ml of 2% (w/v) KMnO₄ in PBS. After 2 h of incubation at room temperature, the fixed samples were washed six times with PBS (20 min for each time), resuspended in 0.5 ml of 2% (w/v) uranyl acetate in PBS and incubated for 4 h at room temperature. Upon the completion of uranyl acetate staining, the cell pellets were washed three times with PBS and then performed an acetone dehydration series, in which cells were left in each step for 20 min at concentrations of 30%, 50%, 70%, 80%, 90% and 100%. Yeast cells were collected and embedded in epoxy resin. After polymerization for 8 h at 70 °C, the embedded samples were sectioned with Leica EM UC7 microtome (Germany). The ultrathin sections (thickness, 70 nm) were examined using a JEM-1400 electron microscope (JEOL CO. Ltd., Japan).

The kidney specimens were fixed with a 4% paraformaldehyde and then incubated with 1% osmium tetroxide (OsO₄) at 4 °C for 1 h. The fixed specimens were dehydrated with gradient alcohols and embedded in Epon. Next, 70–100 nm ultrathin sections were cut with a Leica EM UC 7 microtome and stained with 1% uranyl acetate and lead citrate. Electron micrographs were taken at 30.000× at 80 kV with a JEM 1400 electron microscope (JEOL, Tokyo, Japan) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Microscopic analysis was carried out at the Multiple-access Center for Microscopy of Biological Subjects (Institute of Cytology and Genetics, Novosibirsk, Russia). The number of fenestrae of endotheliocytes of glomerular capillaries and the number of podocyte foot processes were determined for 2 μm of the glomerular basement membrane. Additionally, the thickness of the glomerular membrane and basement membrane of proximal tubular epitheliocytes, as well as the width of podocyte foot processes and slit diaphragm were measured using scale bars.