Cm12 electron microscope

L. mexicana mexicana promastigotes at the density of 10⁵ cells/mL were treated for 72 h with 0.5, 1, or 2× IC₅₀ of eugenol (IC₅₀ = 16.59 µM), or IC₅₀ of amphotericin B (IC₅₀ = 0.25 µM). After centrifugation at 600× g for 10 min, parasites were washed 2 times with phosphate-buffered saline (PBS, pH 7.4) and once with cacodylate buffer 0.1 M (pH 7.4). They were then immobilized on poly-L-lysine-coated coverslips for 5 min at room temperature. After washing 3 times with cacodylate 0.1 M (pH 7.4) to remove the excess of free-floating Leishmania, the coverslips were incubated in 1% glutaraldehyde in cacodylate 0.1 M (pH 7.4) to crosslink the fixed parasites on the poly-L-lysine coating. The parasites were postfixed with 1% osmium tetroxide in a cacodylate buffer for 2 h at 4 °C and washed in water to eliminate traces of the remaining osmium tetroxide, then dehydrated in a graded series of ethanol, critical-point dried, and coated with 10 nm of gold. Samples were observed with a CM12 Philips electron microscope at 80 kV.

Cells exposed to 100 µg/mL TiO₂-NPs for 24 h were washed three times with phosphate saline buffer, fixed with 2.5% glutaraldehyde and post-fixed with OsO₄. They were then dehydrated by immersion in solutions of ethanol with increasing concentration and embedded in Epon. Ultrathin sections were prepared by ultramicrotomy and counterstained with uranyl acetate and lead citrate. These samples were observed using a CM12 Philips electron microscope, operating at 80 kV.

Biofilm cells (IDH781 and EA1002) were grown as described above and fixed overnight using a mixture of 2.5% glutaraldehyde (v/v) and 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C. The glutaraldehyde/paraformaldehyde was removed by vacuum aspiration, the bacterial cells were washed once with PBS, and then dehydrated through a graded ethanol treatment. The cells were removed from the dishes by using propylene oxide. The floating biofilm layers were washed with several changes of propylene oxide, pelleted by low-speed centrifugation, infiltrated and embedded in Epon resin. Ultrathin sections (97 nm) were prepared on an LKB III ultramicrotome, sections were picked up on a 200 mesh copper grid and stained with uranyl acetate and lead citrate. Electron micrographs from a Philips CM12 electron microscope (80 kV; Philips, Eindhoven, The Netherlands) were obtained using an AMT digital camera.

For observation using light microscopy, HeLa WT cells on glass coverslips were rinsed with PBS and then 3 μg of Pt-Dd linked HRP or Bs-Ds linked HRP or free HRP in 50 μL DMEM medium was applied to the cells. After 1 h of incubation at 37°C, the cells were washed three times with PBS to wash out unbound VLPs. Triton X-100 of 0.1% was added for 5 min and the cells were washed again by PBS prior to DAB staining using the substrate solution containing 0.05% 3, 3′-diaminobenzidine (Sigma), 0.05% nickel chloride, and 0.015% H₂O₂ in PBS, pH 7.2 for 5 min at RT in dark storage. The cell staining was mounted on glass slides and observed directly by inverted phase contrast microscope (Leica). The cells incubated by free HRP served as control.
For electron microscopy evaluation, HeLa WT cells in 4 well Permanox® slide (Lab-Tek®) were incubated with 7.5 μg of Pt-Dd linked HRP or free HRP in 100 μL DMEM medium for 1 h at 37°C. For the detection of HRP activity, the cells were incubated for another 1 h in DAB substrate solution as described above. After being rinsed with PBS several times, the cells were fixed in 2% PFA and stained with 2% uranyl acetate. The samples were dehydrated in a graded series of ethanol and propylene oxide and embedded in Spurr's resin. Thin sections were cut with ultramicrotome (Leica), collected onto grids, and examined in a Phillips CM12 electron microscope.

Age-matched non-tg and hSOD1^mus-G37R, -G93A, and -wild-type tg mice (15–17 months of age) received an anesthetic overdose and were perfused transcardially with 2% paraformaldehyde/2% glutaraldehyde. The group sizes were 2 mice per genotype. Tissue samples of left and right biceps femoris were taken from each mouse and processed and embedded in plastic for conventional transmission EM as described (71 (link)). Tissue samples were cut in the transverse plane at 0.5 μm for high-resolution light microscopy at 1000x magnification, and then thin sections were cut and collected on copper grids for EM. These sections were viewed and imaged using a Phillips CM12 electron microscope. Digital electron micrographs from each mouse genotype were used to examine and quantify myofiber sarcomeres, inclusions, mitochondria, and satellite cells in at least twenty images per mouse.