Ultracut r ultramicrotome

LNCaP and C4-2B cells were cultured up to 80% confluence and fixed in 2.5% glutaraldehyde in 0.01 M PBS for 30 min at room temperature (RT). They were then harvested using a scraper, collected into a tube and centrifuged at 800 × g for 5 min at RT. The supernatant was aspirated, while cells were resuspended in warmed 4% gelatin aquatic solution followed by centrifugation at 800 × g for 5 min at RT and cooled on ice. Under a stereoscope the solidified cell pellet with gelatin was extracted, cut into small fragments (1–2 mm³) and transferred into PBS at 4 °C. The cell-gelatin fragments were then dehydrated in graded series of ethyl alcohol, followed by propylene oxide (PO) treatment, infiltrated gradually in a mixture of Epon/Araldite resins diluted in PO, and finally embedded in fresh epoxy resin mixture. Ultrathin sections (70–90 nm thickness) were cut on a Leica Ultracut R ultramicrotome, equipped with a Diatome diamond knife, and mounted onto 200-mesh copper grids. The sections were then counterstained with ethanolic uranyl acetate followed by lead citrate and observed on a Philips 420 transmission electron microscope equipped with an Olympus Megaview G2 CCD camera.

Cells were fixed in a freshly-prepared solution containing 3% formaldehyde (prepared from paraformaldehyde) and 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 min at room temperature (RT). Cells were then harvested using a scraper, collected into a tube and centrifuged at 800 g for 5 min at RT. The supernatant was aspirated, while cells were resuspended in 4% gelatin warmed aquatic solution followed by a spin down at 800 g for 5 min at RT and cooled on ice. Under a stereoscope the solidified cell pellet with gelatin was extracted, cut into small fragments (1–2 mm³) and transferred into 0.1 M phosphate buffer, pH 7.4 at 4 °C. The cell-gelatin fragments were then dehydrated in graded series of ethyl alcohol, followed by propylene oxide (PO) treatment, infiltrated gradually in a mixture of Epon/Araldite resins diluted in PO and finally embedded in fresh epoxy resin mixture. Ultrathin epoxy sections (70-90 nm thickness) were cut on a Leica Ultracut R ultramicrotome, equipped with a Diatome diamond knife, and mounted onto 200-mesh copper grids. Ultrathin sections were observed with a Philips 420 transmission electron microscope and micrographs were taken with an Olympus Megaview G2 CCD camera.

Tissues were fixed in a two-step procedure: initially by immersion in 2% glutaraldehyde, 100 mM sucrose, 0.5 M phosphate buffer, pH 7.3; followed by treatment with 2% osmium tetroxide in 1 M phosphate buffer. After a graded dehydration, the tissues were embedded by infiltration with Epon/Araldite resin (Electron Microscopy Sciences, Fort Washington, PA) and an overnight polymerization at 64°C. Thin sections were cut using a Leica Ultracut R ultramicrotome with a Diatome diamond knife, then mounted on copper grids and contrasted with 4% uranyl acetate and 0.25% lead citrate. The sections were examined in vacuo with electrons accelerated at 60 kV and focused using the magnetic lens of a Philips CM10 transmission electron microscope.