Uranyl acetate and lead citrate

For transmission electron microscopy, fixed brain samples were sliced using a microslicer and fixed in 3% solution of glutaraldehyde (Merck, Darmstadt, Germany) in 0.1 M cacodylate buffer (pH 7.4) (Merck, Darmstadt, Germany) overnight at +40°C. Following osmification, pellets were dehydrated in an ethanol gradient (Galenikaad, Serbia), cleared in propylene oxide (Merck, Germany), and then embedded in Epon (Merck, Germany). Semithin sections were cut using a diamond knife on a Leica Ultracut UCT EM FCS ultramicrotome (Leica Microsystems, Austria), stained with toluidine blue, and analyzed by an Olympus BX41 light microscope (Olympus, Japan). All the slides were photodocumented with an Olympus C-5060 ADU wide-zoom camera and the Olympus DP-soft Image Analyzer program (Olympus GmbH, Germany). The ultrathin sections from chosen representative areas were cut with the same ultramicrotome, collected on copper grids, and stained with uranyl acetate and lead citrate (SERVA Electrophoresis, Germany). The sections were examined by FEI Morgagni 268D transmission electron microscopy (FEI Europe, Netherlands) and photographed with a Mega View III Soft Imaging System digital camera (Olympus Soft Imaging Solutions, Germany).

Gonads samples for TEM examination were fixed in 2.5% glutaraldehyde (Serva Electrophoresis, Heidelberg, Germany). After 24 hours, the specimens were washed in the cacodylate or phosphate buffers (0.1 M, pH 7.4, Serva) and postfixed for 1 h in 1% osmium tetroxide (Serva). Subsequently, the specimens were dehydrated in ethyl alcohol and twice in pure acetone (Chempur, Poland). Afterwards samples were embedded in epoxy resin (Epon 812, Serva). Epon blocks were cut into semithin (600 nm thick) sections and stained with toluidine blue (Serva). Finally, ultrathin (50 nm thick) sections were prepared using an ultramicrotomes Power Tome XL (RMC, Tucson, USA) and Reichert Ultracut E. Ultrathin sections were counterstained with uranyl acetate and lead citrate (Serva), and examined in a TEM JEM-1011 (JEOL,Tokyo, Japan) or Zeiss EM 900. Digital micrographs were collected with the use of TEM imaging platform iTEM1233 equipped with the Morada Camera (Olympus, Münster, Germany).