Em910 electron microscope

Newborn Mpdz‐deficient and wild‐type littermates (n = 3 for each age and genotype) were euthanized and perfused with PBS and subsequently with 4% glutaraldehyde and 4% formaldehyde in 0.1 M cacodylate buffer through the left ventricle of the heart. Dissected brains were fixed in 2.0% glutaraldehyde in 0.05 M cacodylate buffer for 2 h and stained with 1% OsO₄ in cacodylate buffer for another 2 h followed by contrasting in 0.5% uranyl‐acetate. After dehydration in an ascending series of ethanol and propylene oxide, the samples were flat‐embedded in Epon (Serva, Germany). Using an ultramicrotome (Ultracut, Leica, Bensheim, Germany), 0.5‐μm‐ and 50‐nm‐thin sections were cut. Ultrathin sections were stained with 2% uranyl‐acetate for 15 min and contrasted in lead citrate for 5 min, mounted on copper grids, and finally analyzed with a Zeiss‐EM910 electron microscope.
For scanning electron microscopy, glutaraldehyde‐fixed brains were dehydrated, transferred into hexamethyldisilazane, and slowly air‐dried. Samples were cut and coated with gold using the agar sputter coater (Agar Scientific, England). Images were taken using the Hitachi S4500 and analyzed with the digital image processing 2.6 software (Point Electronic, Halle).

Caco-2 cells were seeded at 7.5 × 10⁴ cells/cm² on transwell inserts of 24 well plates (polyester membrane, pore size 0.4 µm; Corning Life Sciences, Amsterdam, Netherlands) and cultured until the cells formed a confluent monolayer and further 7, 14 or 21 days. For transmission electron microscopical analysis, the cells were treated according to the protocol of Bye et al. [32 (link)]. They were washed once with 0.1 M sodium cacodylate (pH 7.4) and fixed with a solution containing osmium tetroxide and glutaraldehyde as fixatives (0.5% osmium tetroxide (Polysciences Europe GmbH, Eppelheim, Germany), 2.5% glutaraldehyde, 10 mM potassium hexacyanoferrate(II) (both from Sigma-Aldrich/Fluka, Taufkirchen, Germany), 0.05 M sodium cacodylate (Carl Roth, Karlsruhe, Germany)). After fixation the cells were washed once again with sodium cacodylate, dehydrated over a serial dilution with ethanol and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined with a Zeiss EM910 electron microscope; images were taken at 2,500× and 12,500× magnification.

Dissected pieces of PSI tissue of 2-3 mm³ from 8- to 12-week-old mice were fixed by immersion in 4% (w/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer for 2 h at room temperature. Samples were post-fixed with 1% (v/v) osmium tetroxide for 3 h at room temperature, dehydrated in a graded series of ethanol, and embedded in PolyBed 812 resin (Polysciences). Ultrathin sections (60-80 nm) were stained with uranyl acetate and lead citrate, and examined at 80 kV with a Zeiss EM 910 electron microscope. Images were acquired with a Quemesa CCD camera using iTEM software (EMSIS).

Cell-containing microfibers were fixed in glutaraldehyde 2.5% buffered solution and osmium tetroxide 2% buffered solution and dehydrated through an ethanol gradient; samples were araldite embedded (ACM Fluka Sigma-Aldrich Co., St. Louis, MO, USA) and the ultra-thin sections of a selected area were contrasted with uranyl acetate lead citrate and observed at transmission electron microscopy (TEM; ZEISS EM 910 electron microscope; Zeiss, Oberkochen, Germany). For toluidine staining, 5 µm sections from the same specimens were obtained with a glass blade. Sections were stained with toluidine blue, mounted in glycerol, and observed using a Leitz microscope.

Mice were fixed by perfusion with 4% (w/v) formaldehyde in 0.1 M phosphate buffer and hearts were dissected to 1–2 mm³ cubes. Tissue blocks and HUVEC cells were fixed in phosphate buffered 2.5% (v/v) glutaraldehyde for 1–2 hours. Cells were pelleted, stabilized by 10% (w/v) gelatin and further processed as 1–2 mm³ cubes. Additional membrane contrast was added first by 1% tannic acid in 0.1 M cacodylate buffer and second by aqueous 2% uranylacetate. After 1% (v/v) osmium tetroxide treatment, tissue and cells cubes were dehydrated in a graded series of ethanol and embedded in the PolyBed® 812 resin (Polysciences Europe GmbH), ultrathin sections (60–80 nm) were cut (Leica microsystems) and uranyl acetate and lead citrate staining was performed. Samples were examined at 80 kV with a Zeiss EM 910 electron microscope (Zeiss), and image acquisition was performed with a Quemesa CDD camera and the iTEM software (Emsis GmbH).