Cm24 8gf

Ultrathin LR-White embedded sections, collected on Formvar carbon-coated nickel grids, were incubated in drops of 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.02 M glycine and normal goat serum at room temperature for 30 min [38 (link)]. Sections were then incubated overnight with a rabbit monoclonal anti-vimentin antibody (clone V9; Ventana, Tucson, AZ, USA; pre-diluted) at 4°C. After several washes with PBS + 0.1% BSA, grids were incubated with a 20 nm secondary antibody-gold particle complex (Agar Scientific, Stansted Essex CM24 8GF United Kingdom) at 1:10 diluted in PBS 0.1% BSA for 2 h at room temperature. After immunolabeling, sections were washed with PBS + 0.1% BSA, washed in distilled water, dried, and counterstained with uranyl acetate. All sections were examined with a Hitachi 7100 FA electron microscope.

Small fragment of prostate tissue (1 mm³) was fixed in 4% paraformaldehyde and postfixed in 2% osmium tetroxide. Then, the sample was dehydrated in alcohol and infiltrated with propylene oxide before being embedded in Epon (Agar Scientific, Stansted CM24 8GF, Essex, United Kingdom) [15 (link)]. Eighty-micrometer ultrathin sections were cut by ultramicrotome and mounted on copper grids. All samples were examined with a transmission electron microscope (Model JEM-1400, JEOL) [16 (link)–18 (link)].
For EDX microanalysis, 80 µm ultrathin sections were mounted on copper grids. Hydroxyapatite crystals were identified by EDX detector (Thermo Scientific, Waltham, MA, USA) at an acceleration voltage of 75KeV and magnification of 12.000 [16 (link)–18 (link)]”.

RTT patients enrolled in the study were analyzed by TEM. The figures presented are relative to the analyses performed on the patients carrying the R255X MeCP2 mutation. Additional patients were analyzed and are not included in the results. They carry the following MeCP2 mutations: T158M (2 patients), R270X (2 patients), P384 (early truncation), Exon-3 complete deletion and Exon-4 partial deletion, A168X, R294X, T327fs, plus one with unknown mutation. Blood samples were fixed with an equal volume of buffered Karnosky’s fixative. The samples were then dehydrated through an alcohol series and then infiltrated and embedded in a liquid EPON resin (Agar Scientific, Stansted Essex CM24 8GF United Kingdom), for morphological and ultrastructural analysis. After embedding, the resin blocks were then thin sectioned; sections of 50–70 nm thickness were collected on metal mesh ‘grids’ and stained with heavy metal solutions before observation in the TEM. All sections were examined by a Hitachi 7100 FA electron microscope.