902 electron microscope

BMDCs were infected with TgRH YFP SAG1-OVA at MOI 1 during 24 h at 37°C. Then, cells were washed with ultrapure PBS three times, and fixed with 2.5% glutaraldehyde during 1 h at 4°C. Cells were washed again three times with ultrapure PBS (5 min at 4°C each), and incubated in 1% osmium tetroxide/PBS for 2 h at RT. Then they were dehydrated sequentially with increasing concentrations of ice-cold acetone and three times with 100% acetone for 15 min at RT. Cells were infiltrated in 1:1 acetone:EPON overnight at RT and finally embedded in fresh pure resin overnight at RT. Thin sections (60–80 nm) were cut with a diamond knife (Diatome, Washington, DC) on a Leica Ultracut R ultramicrotome and collected on 200-mesh copper grids. Grids were observed and photographed in a Zeiss 902 electron microscope at 50 kV.

Eyes were immersed in 2% glutaraldehyde in 0.1M phosphate buffer (PB), at pH 7.4 and 4°C for 5h. After being washed in 0.1M PB, the wall of the posterior segment of the eyes (including the choroid, RPE, and neurosensorial retina) was diced into small pieces. These fragments were post-fixed in 1% osmium tetroxide in 0.1M PB for 2h at 4°C. The tissues were then dehydrated in graded acetone and embedded in araldite. The semi-thin sections (0.5μm) were stained with toluidine blue, and after selection the blocks were further trimmed for ultramicrotomy (Reichert OM-V3 ultramicrotome, Leica, Germany). The thin sections, treated with 2% uranyl acetate in water and lead citrate for contrast, were examined by transmission electron microscopy (TEM; Zeiss 902 electron microscope) to study the RPE and the neurosensory retina.

Cell cultures were fixed with 0.05% glutaraldehyde and 4% paraformaldehyde in 0.2 M Pipes buffer for 30 min at room temperature then at 4 °C for 1H. Cells were then incubated in phosphate-buffered saline containing 10% fetal calf serum, scraped, and centrifuged at 15,000 g. Pellets were soaked overnight in phosphate-buffered saline containing 2.3 M sucrose and 20% polyvinylpyrrolidone. Cells were rapidly frozen in liquid nitrogen. Frozen ultrathin sections were made with a cryo-ultramicrotome (Leica EM UCT) at a thickness of 85 nm with a cryochamber (Leica EM FCS). The sections were picked up on formvar-carbon-coated nickel grids. After a 30 min saturation in 2% bovine serum albumin, immunolabeling was carried out using Tau5, an anti-total Tau antibody, and an antibody recognizing H3K9me3. Tau5 labeling was revealed with a goat anti-mouse IgG gold conjugate (12 nm in diameter) (Jackson ImmunoResearch Laboratories) and H3K9me3 labeling was revealed with a goat anti-rabbit IgG gold conjugate (6 nm in diameter) (Jackson ImmunoResearch Laboratories). Negative staining of the ultrathin sections was carried out using 0.4% uranyl acetate and 1.8% methyl cellulose. Labeling was observed under a Zeiss 902 electron microscope.