Morgagni tem

If not otherwise mentioned, incubations were at RT. Samples were fixed in the culture dishes 30 min in 150 mM HEPES, pH 7.35, containing 1.5% formaldehyde and 1.5% glutaraldehyde. After immobilization in 2% agarose, samples were incubated 2 hr in an aqueous solution of 1% OsO₄ containing 1.5% hexacyanoferrat II, washed in water and stored in 1% aqueous uranyl acetate overnight at 4 °C. After washing in water and dehydration in acetone, samples were embedded in Epon. 60 nm ultrathin sections were mounted on formvar-coated copper grids, poststained with uranyl acetate and lead citrate⁴⁵ and observed in a Morgagni TEM (FEI). Images were recorded using a side mounted Veleta CCD camera. Two independent sets of osteoclasts differentiation were used for transmission electron microscopy experiments. Both sets have shown the same results.

Liposomes were pelleted and resuspended in a tenfold volume of fixation buffer (150 mM HEPES, pH 7.35, containing 1.5% formaldehyde and 1.5% glutaraldehyde) for 30 min at RT and overnight at 4 °C. Liposomes were postfixed in 1% osmium tetroxide 2 h at RT and 4% uranyl acetate at 4 °C overnight. After dehydration in acetone, samples were embedded in EPON. 50-nm-thick sections were post-stained with 4% uranyl acetate and lead citrate [14 ] and observed in a Morgagni TEM (FEI), operated in the bright field mode. Images were recorded at 80 kV using a 2 K side mounted Veleta CCD camera, binned to 1 K. The number of unilamellar vs. bilamellar liposomes in liposomes without cholesterol was determined by inspection of a total of 793 liposomes. 12% were found to possess two rather than one membrane. Liposomes with cholesterol exhibited unilamellar liposomes only.

At 2 h, 24 h and 48 h post-treatment, AT samples were collected and fixed with a 2.5% glutaraldehyde and 2% paraformaldehyde in PBS for 2 h at 4 °C. The samples were then rinsed with PBS, post-fixed with 1% OsO₄ and 1.5% K₄Fe(CN)₆ for 2 h at 4 °C, dehydrated in acetone and embedded in Epon resin. Ultrathin sections were stained with Reynolds lead citrate and observed in a Philips Morgagni TEM (FEI Company) equipped with a Megaview III camera. At least three AT samples per animal were analysed.
The area of small lipid droplets extruding from the central one was measured in a total of 500 µm² of cytoplasm per experimental condition (X7′100) using the ImageJ software (NIH), and their total area was calculated and expressed as percentage of the measured cytoplasmic area.
A morphometric analysis was carried out also on 30 randomly-chosen mitochondria (X28′000) per control, O₂-, 10 µg- or 20 µg O₃-treated samples: the mitochondrial area and the ratio between inner and outer membrane (estimating the extension of cristae independently of the mitochondrial size) were assessed. The means ± SE were calculated and a statistical comparison was performed as described below.

Protein samples were diluted to 0.1 to 0.15 mg/mL and applied to freshly glow discharged (60 s, 5 mA, PELCO easiGlow) 200 mesh gold formvar/carbon square mesh grids (EMS, Cat # FCF200-AU-EC). After 60 s, the samples were blotted of the grids using filter paper. Grids were then washed with a 5 μL drop of distilled water, followed by two rounds of 5 s and then 30 s staining with 5 μL drops of 0.75% uranyl formate. TEM micrographs were recoreded using an FEI Morgagni TEM operating at 100 kV located at the Michigan University Life Sciences Institute.

See description in (Jarriault et al., 2008 (link)) (SI text). Ten microns of serial ultrathin sections (50–70 nm) of the rectal area were collected and contrasted in lead citrate and uranyl acetate before imaging with a SiS Megaview 3 CCD camera mounted on a FEI Morgagni TEM operated at 70 kV.