Durcupan

Epidydimal sperm samples were centrifuged and the resulting pellet embedded in 2% agarose for transmission electron microscopy. Thereafter, cells/tissue samples were contrasted using 1% osmium tetroxide in PBS for 45 min at room temperature and 1% uranyl acetate (45 min at room temperature in 70% ethanol). Agar blocks were dehydrated and embedded in epoxy resin (Durcupan, Sigma-Aldrich, Germany). Ultrathin sections were prepared using a Leica UC6 ultra-microtome and analysed using a Philipps CM100 transmission electron microscope.

The cells were processed for transmission electron microscopy as described previously [23 (link)]. Briefly, the cells were fixed in 3% glutaraldehyde (in 0.1 M cacodylate buffer, pH 7.2; Sigma) for 3 h at room temperature. Subsequently, the cells were washed in cacodylate buffer (0.1 M, pH 7.2) and post-fixed in 1% OsO4 (in 0.1 M cacodylate buffer, pH 7.2; Sigma) for 1 h at room temperature. After the rinsing procedure, the cells were dehydrated in graded alcohols (50%, 75%, 96%, and 100%), clarified in propylene oxide, and embedded in a mixture of Epon 812 and Durcupan (Sigma; polymerization for 3 days at 60 °C). Toluidine blue was used to stain the semithin sections. Ultrathin sections were cut on Ultrotome Nova (LKB, Vienna, Austria). These sections were then collected onto formvar carbon-coated copper grids, counterstained with uranyl acetate and lead citrate, and examined under a JEOL JEM-1400Plus transmission electron microscope (at 120 kV, JEOL, Tokyo, Japan). The images were taken with the integrated 8Mpix CCD camera and processed further using the software TEM Center (Ver. 1.7.3.1537, JEOL, Tokyo, Japan).

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.

Dissected proventriculi with garland nephrocytes attached were fixed in 3.2% paraformaldehyde, 0.5% glutaraldehyde, 1% sucrose, and 0.028% CaCl₂ in 0.1 N sodium cacodylate, pH 7.4 for overnight at 4°C, postfixed in 0.5% osmium tetroxide for 1 hr and in half-saturated aqueous uranyl acetate for 30 min, dehydrated in a graded series of ethanol and embedded into Durcupan (Fluka - Sigma Aldrich) according to the manufacturer’s recommendations. 70-nm sections were stained in Reynold’s lead citrate and viewed on a transmission electron microscope (JEM-1011; JEOL, Tokyo, Japan) equipped with a digital camera (Morada; Olympus) using iTEM software (Olympus).

Four C57BL/6 mice of either sex with an age between P23–P28 were sacrificed, followed by transcardial perfusion with saline and consecutively a fixative containing 4% paraformaldehyde and 2% glutaraldehyde in phosphate-buffered saline (PBS). After removal of the brain, the tissue was allowed to post-fix over night at 4°C and sagittal sections of the cerebellum were prepared at a thickness of 60 µm using a Leica microtome (Leica Microsystems, Wetzlar, Germany). The sections were stained in 0.5% osmium tetroxide in PBS for 30 min followed by dehydration in graded alcohol and another staining step with 1% uranyl acetate in 70% ethanol. After further dehydration, the tissue was embedded in durcupan (Sigma-Aldrich), which was allowed to polymerize for 48 hr at 56°C between coated microscope slides and cover glasses. Regions of interest were identified by light microscopy, cut and transferred onto blocks of durcupan to obtain ultra-thin sections using an Ultramicrotome (Leica Microsystems). Ultra-thin sections were transferred onto formvar-coated copper grids and stained with lead citrate. Ultrastructural analysis was performed using a Zeiss SIGMA electron microscope (Zeiss NTS, Oberkochen, Germany) equipped with a STEM detector and ATLAS software.