Fat bodies were dissected out and fixed in Karnovsky mixture containing 4% formaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer for 2 h. Then, the fixed tissues were washed and treated with 1% OsO4 for 1 h. After dehydration with a series of cold-graded acetone solutions, the tissues were embedded in Embed 812 resin. Following a 48-h polymerization at 60°C, semithin sections of 200 nm were obtained using a JEOL microtome equipped with a diamond blade and were stained with toluidine blue for high-resolution light microscopy analysis. For transmission electron microscopy (TEM), 90-nm ultrathin sections (JEOL JUM-7 ultramicrotome) were placed on grids which were later post-stained with uranyl acetate/lead citrate, examined using a Zeiss Leo 906-E electron microscope (Oberkochen, Germany), and photographed with a Megaview III camera (Olympus, Center Valley, PA). The size of the lipid droplets was determined by measuring their diameter in a minimum of 25 trophocytes from ultrathin sections of each experimental condition.
Microtome
Manufactured by JEOL
Sourced in Japan
The JEOL microtome is a precision instrument used for sectioning samples for microscopic analysis. It allows for the cutting of thin, uniform slices from solid specimens, such as biological tissues or materials, to prepare them for examination under a microscope. The microtome facilitates the preparation of high-quality samples by providing precise control over the thickness of the sections, enabling detailed observation and analysis.
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Lab products found in correlation
2 protocols using microtome
1
Ultrastructural Analysis of Insect Fat Bodies
2
TEM Imaging of Insect Hemocytes
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For TEM assays, hemolymph collected from 10 insects was pooled and centrifuged at 500× g for 10 min at room temperature [31 (link),32 (link)] to enrich the preparation with hemocytes without inducing visible cell damage. The supernatant was discarded and the pellet was suspended and fixed in Karnovsky mixture containing 4% formaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer for 2 h and processed as previously described [33 (link)]. Fixed cells were washed and treated with 1% OsO4, dehydrated in a series of acetone solutions, and embedded in Embed 812 resin. After a 48-h polymerization stage, 200 nm semithin sections obtained using a Jeol microtome (Tokyo, Japan) were stained with toluidine blue for high-resolution light microscopy analysis. Finally, 90-nm ultrathin sections obtained in a JUM-7 ultramicrotome (Jeol, Tokyo, Japan) were placed on grids, post-stained with uranyl acetate/lead citrate solutions, examined in an electron microscope (Zeiss Leo 906-E, Oberkochen, Germany), and photographed with a Megaview III camera (Olympus, Center Valley, PA, USA).
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