Leo912 electron microscope

After thawing, six pieces of central and peripheral retina were excised and fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 m phosphate buffer with pH 7.4. After thorough rinsing in buffer three blocks were postfixed in osmium tetroxide (2%) in the same buffer for 2.5 h. The blocks were rinsed again, dehydrated in a graded series of alcohol and embedded in Epon.
For light microscopy 1–2 μm sections of non-osmicated tissue were stained according to Richardson40 (link) and photographed with a digital camera mounted on an Axioscope (Carl Zeiss Microscopy GmBH, Jena, Germany). For standard electron microscopy 60 nm sections of osmicated tissue were prepared, the contrast of most sections was enhanced with lead citrate and viewed with a LEO 912 electron microscope (Carl Zeiss NTS, Jena, Germany).
The electron microscope was equipped with an omega filter which allowed sections to be irradiated with near monochromatic electron beams. Using electron energy loss spectroscopy (TEM EELS; acceleration voltage 80 kV) allowed the visualisation of the amount and localisation of the elements phosphorus, sulphur and calcium in tissue sections, indicative of proteins and/or nucleic acids. For this analysis 30 nm sections of non-osmicated tissue were used and image processing and subtraction performed with analySIS 3.0 software (Soft Imaging Systems GmbH, Münster, Germany).

After brief treatment with ATP, CHME-5 cell monolayers were detached and sedimented by centrifugation (1,000 rpm; 5 min); the pellets were then fixed with 4% paraformaldheyde-2% glutaraldheyde in PBS (for 30 min), post-fixed with 1% OsO₄ (1 h), then washed and embedded in Epon. Conventional thin sections were collected on uncoated grids, stained with uranyl acetate and lead citrate, and examined in a Leo 912 electron microscope (Zeiss).
Microvesicles were observed at TEM after being contrasted by negative staining: cell media were fractionated by differential ultracentrifugation, the resulting pellets resuspended in 20 µl of PBS and adsorbed to 400-mesh formvar/carbon coated grid for 10 min at RT. Adherent vesicles were stained with uranlyl acetate and immediately observed at the electron microscope.