Leica em uc6 microtome

Specimens stored in 70% ethanol were dehydrated in a graded ethanol series and embedded for serial sectioning. Early yolk - rich stages (stages 19 to 24) were embedded in epoxy resin (Epon 812, TAAB Ltd., Rome, Italy) and sectioned into 2-μm semi-thin serial sections using a glass knife and a Leica EM UC6 microtome (Leica Microsystems, Milan, Italy) in order to prevent breaking of the brittle tissue. Sections were stained with toluidine blue for 20 s at 100°C. Later stages (stages 26 to 30) were embedded in paraffin (stages 26 to 30), sectioned into 4-μm thin sections using a Leica RM2125 microtome (Leica Microsystems, Milan, Italy) and stained with Masson’s trichrome stain in order to identify mature muscle fibers. For each stage four embryos were embedded, sectioned, and analyzed.

Pieces of the quadriceps muscle (two samples of vastus lateralis per group) were fixed in 0.1 M of PBS (pH 7.4) solution with the addition of 2.5% glutaraldehyde for 2 h. The sample was then fixed in PBS solution supplemented with 1% osmic acid and dehydrated by increasing the alcohol concentration. At the next stage, the samples were encapsulated in Epon 812 resin. Ultrathin sections that were 70–75 nm thick were made using a Leica EM UC6 microtome (Leica Microsystems, Wetzlar, Germany). The sections were stained with uranyl acetate and lead citrate. The skeletal muscle samples were visualized using a JEM-100B electron microscope (JEOL, Tokyo, Japan). An Epson V700 scanner was used to digitize the negatives, and the resulting images were analyzed using Image Tool 3.0 software.

For the electron microscopy study, samples of the liver were taken from the edge of the left lateral lobe and fixed for 2 h in a 2.5% glutaraldehyde solution in 0.1 M phosphate-buffered saline (PBS, pH 7.4). After washing with the buffer, the tissue was fixed for 2 h with a 1% solution of osmium acid in PBS and dehydrated by increasing concentrations of alcohols. The resulting samples were encapsulated in Epon 812 resin. Ultrathin sections (70–75 nm) were prepared on a Leica EM UC6 microtome (Leica Microsystems, Wetzlar, Germany) and stained with uranyl acetate and lead citrate. The preparations were examined and photographed using a JEM-100B electron microscope (JEOL Ltd., Tokyo, Japan). Ultrastructural analysis was performed using negative images digitized with an Epson V700 scanner. The morphometric analysis of images was conducted on photographic negatives using the Image Tool software.

For the electron microscopy examination, pieces of the skeletal muscle (quadriceps, two samples in each experimental group) were taken and fixed for 2 h in 0.1 M phosphate-buffered saline (PBS, pH 7.4) supplemented with 2.5% glutaraldehyde. Further, the tissue was fixed in PBS containing 1% osmic acid, and water was removed by increasing the alcohol concentration. The resulting skeletal muscle samples were encapsulated in Epon 812 resin. A Leica EM UC6 microtome (Leica Microsystems, Wetzlar, Germany) was used to prepare ultrathin sections (70–75 nm) and then stained using lead citrate and uranyl acetate. The samples were evaluated and imaged using a JEM-100B electron microscope (JEOL, Tokyo, Japan). Negative images digitized with an Epson V700 scanner were used for morphometric analysis with the Image Tool 3.0 software, which included an assessment of the size of sarcomeres and mitochondria.

Immunogold staining was conducted at the EM Facility of the University of British Columbia (Vancouver, BC, Canada). In brief, BCG-infected macrophages were fixed with 4% paraformaldehyde, embedded in 4% low melting point agarose and dehydrated in ethanol. Samples were then transferred to LR White resin. After polymerization at 50°C, 60 nm sections were cut with a Leica EM UC6 microtome (Leica Microsystems, Switzerland) and collected on nickel grids. Samples were labeled with avidin antibodies then gold-conjugated F(ab')₂ of ultra-small goat-anti-rabbit IgG. Sections were then washed in distilled water, stained in 2% glutaraldehyde, washed again, air dried and examined with Tecnai G2 200kV electron microscope (FEI Company, Hillsboro, OR).

Electron microscopic studies were carried out using pieces of liver taken immediately after decapitation of animals. The tissue was fixed in a 2.5% glutaraldehyde solution in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 2–3 h, followed by a 2 h incubation in a 1% solution of osmic acid. Subsequently, the samples were dehydrated in increasing concentrations of alcohol and in absolute acetone, and encapsulated in Epon 812 epoxy resin. Ultrathin (60–75 nm) sections were prepared on a Leica EM UC6 microtome (Leica microsystems, Wetzlar, Germany) and stained with uranyl acetate and lead citrate. The preparations were examined and photographed by a Tecnai Osiris FEI (USA). The morphometric analysis of images was carried out using ImageJ 1.52n software; 35–50 electron micrographs for each individual animal (n = 2–3) were studied. The different stages of mitochondrial damage are presented: Score 1: healthy mitochondria (well-defined, intact, organized membranes and cristae); Score 2: swollen mitochondria, a slight decrease in the electron density of the matrix and irregular cristae; Score 3: swollen mitochondria with MLBs; Score 4: matrix vacuolization, cristae are almost not defined; Score 5: damaged mitochondria with vacuolized matrix and MLBs.