Ultracut r

The samples collected during surgery were taken for primary fixation at 4 °C
for 24 hours in 2.5% glutaraldehyde containing 0.1 M phosphate buffer, and
then washed 3 times for 15 minutes with phosphate buffer. The blocks
obtained were cut at 700 nm thickness with an ultramicrotome (Leica Ultracut
R) and stained with toluidine blue. After determining the areas to be
displayed in transmission electron microscopic (TEM), every subject was
scored according to the criteria shown in Table 1^{[16 (link)]}.

The second internode, 10th internode, and 20th internode (counting from the tip) were taken from 4-month-old wild-type and 35S::PtoCYCD3;3 plants. The samples were fixed in FAA (70% ethanol: glacial acetic acid: formaldehyde: glycerol = 18:1:1:1) for 24 h at room temperature and then dehydrated through a graded ethanol series (70%, 80%, 90%, and 100% ethanol; 20 min each step). Ethanol was replaced with ethanol: acetone (1:1) for 20 min and then with pure acetone for 20 min two times. Afterward, the samples were sequentially infiltrated with acetone: resin (2:1) for 3 h and then with acetone: resin (1:1) and acetone: resin (1:3) for 3 h. The samples were replaced with fresh resin for 12 h. After the resin replacement was repeated two times, stem segments were embedded into the resin for 24 h at 60 °C. One µm sections were obtained with a microtome (Leica ultracut R, Germany). The sections were floated on the water, heat-fixed to glass slides, stained with 0.05% toluidine blue, and observed under a Leica DM4000B light microscope. Data were measured on Image J. We measured five times in different quadrant sectors of the cross-sections about the width of cambium-derived tissues. All statistical analyses were performed using SPSS v16.0. Independent-Samples T-test was used.

RAW macrophages and H9C2 cells were infected with trypomastigotes of the Y strain with 10 parasites per cell (MOI 10) for 24 h. They were then washed to eliminate the parasites that did not cause infection, and a fresh medium was added with or without 10 µM of UA for 24 additional hours. After that, cells were fixed with 2.5% glutaraldehyde (Pelco International, Fresno, CA, USA) in PBS at 10°C and processed by the Servicio de Preparación de Muestras de Microscopía Electrónica, STAN 3371IHEM-CONICET.
Briefly, each sample was washed in the same buffer, post-fixed in 1% OsO for 1 h at room temperature, dehydrated in a graded acetone solution series, and embedded in low viscosity epoxy resin (Pelco International). Then, ultrathin sections with interference color gray were cut by ultramicrotome Leica Ultracut R, mounted on grids, and stained with uranyl acetate and lead citrate (Reynolds 1963 (link)). Grids were examined under a Zeiss 900 electron microscope, with a Gatan digital camera (model Orius SC 1000).