900 electron microscope

C. neoformans, FLC sensitive, and resistant C. albicans strains were treated with subinhibitory concentrations of the EO (625 μg/mL for all microorganisms) and geraniol (38 μg/mL for C. neoformans and 76 μg/mL for both C. albicans strains) for 48 h at 37°C. The yeasts were washed in PBS, pH 7.2, and fixed in a solution of 2.5% glutaraldehyde and 4% formaldehyde in 0.1 M cacodylate buffer, pH 7.2, for 1 h at room temperature. Next, the yeasts were postfixed for 2 h in 1% osmium tetroxide containing 1.25% potassium ferrocyanide and 5 mM CaCl₂ in cacodylate buffer, pH 7.2. The yeasts were then washed in the same buffer, dehydrated with increasing ethanol concentrations (30, 50, 70, 90, and 100% and ultradry ethanol), with the cells remaining in each concentration for 30 min, and then embedded in Spurr resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and images were obtained using a Zeiss 900 electron microscope equipped with a CCD Camera (mega view III model, Soft Image System, Germany). The images were processed with iTEM software (Soft Image System, Germany).

Promastigotes of Leishmania and epimastigotes of T. cruzi were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and postfixed in a solution containing 1% OsO4, 1.25% potassium ferrocyanide and 0.1 M cacodylate buffer, pH 7.2. For transmission electron microscopy, cells were dehydrated in acetone and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under a Zeiss 900 electron microscope^{16 (link),18 (link)}. Several 2D thin sections were analyzed for each JVPH3 treatment group and untreated parasites. There were around at least 15 to 20 cells in each section. For scanning electron microscopy, promastigotes were dehydrated in ethanol, critical point-dried in CO₂, mounted on stubs, sputtered with a thin gold layer and observed under a FEI Quanta 250 scanning electron microscope^{6 (link),18 (link)}. At least 100 cells were observed for untreated and JVPH3 treated parasites.

HeLa cells grown in T-25 mL flasks were infected with C. trachomatis L2 (Ct; MOI of 5) and treated with 5 μM iAkt from 2 h pi until its fixation at 24 h pi. Infected cell monolayers were fixed with 2% glutaraldehyde/PBS for 1 h at 37°C. Then, cells were removed with 1% gelatin/PBS, gently centrifuged (15 min at 1200 rpm) and washed three times with PBS. After that, cells were incubated with Osmium tetroxide/Potassium ferricyanide/PBS (1:1:1) for 90 min. Samples were dehydrated using increasing acetone series and embedded in Spurr’s resin (Ted Pella Inc., United States). Thin sections were cut with an ultramicrotome (Leica ultracut R, Austria) and stained with 1% uranyl acetate and Reynold’s lead citrate (Ted Pella Inc., United States) before they were observed with a Zeiss 900 electron microscope (Zeiss, Germany). Images were processed using Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA, United States).

Epimastigotes were exposed to the first period of metacyclogenesis in Diamond (control) or TAU (starved) medium, during 2 h at 37°C and then fixed and processed by Electron Microscopy. Briefly, parasites were fixed with 2% glutaraldehyde (Ted Pella) in PBS for 2 h at 4°C, washed three times with PBS pH 7.2 and subsequently treated with 1% osmium tetroxide (Ted Pella) for 2 h at 4°C. In a next step, parasites were washed again with PBS and sequentially dehydrated in solutions with increasing concentrations of acetone. Finally, samples were included in the epoxy resin (Spurr) and ultrathin sections in an ultramicrotome Leica Ultracut R were performed. Sections were contrasted with uranyl acetate / acetone for 3 min, washed with distilled water and colored with lead citrate for 2 min before observation with the Zeiss 900 electron microscope.

After 48 h of culture in serum-free medium, H-MSCs and E-MSCs were collected by centrifugation, washed in PBS, and fixed for 24 h at 4°C in 2.5% glutaraldehyde in 0.1 M cacodylate buffer. After fixation, MSCs were washed with cacodylate buffer and post-fixed in a solution containing 1% OsO₄, 1.25% potassium ferrocyanide, 5 mM CaCl₂, and 0.1 M cacodylate buffer for 30 min. MSCs were then washed in the same buffer. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under a Zeiss 900 electron microscope (Carl Zeiss, Jena, Germany).

First, control and treated promastigotes and intracellular amastigotes were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 h at room temperature. Second, the samples were postfixed in a solution containing 1% OsO₄, 1.25% potassium ferrocyanide, and 5 mM CaCl₂ in 0.1 M cacodylate buffer (pH 7.2) for 30 min. For scanning electron microscopy, promastigotes were dehydrated in ethanol (30, 50, 70, 90, and 100%) and critical point-dried in CO₂. After that, samples were sputtered with a thin gold layer and observed under a FEI Quanta 250 scanning electron microscope. For transmission electron microscopy, cells were dehydrated in acetone and embedded in epoxy resin. After that, ultrathin sections were stained with uranyl acetate and lead citrate and observed under a Zeiss 900 electron microscope.