B. subtilis cells were fixed in buffered 2.5% glutaraldehyde and 4% paraformaldehyde. They were then washed three times with physiological saline to remove excess fixative, fixed in unbuffered 1% osmium tetroxide, and washed with physiological saline. After dehydration, the bacteria were treated with pure acetone for 20 min, mixed solution of embedding agent and acetone (v/v = 1/1) for 1 h, pure acetone for 20 min, a mixed solution of embedding agent and acetone (v/v = 3/1) for 3 h, and pure embedding agent overnight. After heating at 70 °C overnight, the samples were obtained and sliced into 70–90 nm sections by a LEICA EM UC7 ultra-thin microtome. The sections were stained with lead citrate solution, uranyl acetate, and 50% ethanol saturated solution for 5–10 min, respectively, and then viewed under a transmission electron microscope.
Em uc7 ultra thin microtome
Manufactured by Leica
The EM UC7 ultra-thin microtome is a laboratory equipment designed for cutting thin sections of materials for microscopic examination. It features a motorized, high-precision cutting system that can produce sections with thicknesses ranging from 15 nanometers to 100 micrometers. The EM UC7 is suitable for a variety of materials, including biological samples and hard materials.
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5 protocols using em uc7 ultra thin microtome
1
Transmission Electron Microscopy of Bacillus subtilis
2
Ultrastructural Analysis of Mouse Testes
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After the mice were anesthetized, 50 ml of fixative solution (2% paraformaldehyde and 2% glutaraldehyde) was perfused into the heart within 1 h, and the testes were then removed and fixed in the fixative solution for 4 h. After washed with 0.1 M PB buffer (80 mM Na2HPO4 and 25 mM NaH2PO4) for 3 times, the tissues were immersed in 1% osmium acid for 2 h. Through the acetone gradient dehydration and immersion of acetone: epoxy resin (1:1), the tissues were embedded, and placed at 60 °C for 24 h. The sections (70 nm) were sliced via the Leica EM UC7 ultra-thin microtome, and stained with uranyl acetate and lead citrate. Images were captured and analyzed with JEM-1400 electron microscope and Gatan digital camera.
3
Transmission Electron Microscopy Sample Preparation
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Cells were harvested, and the supernatant was discarded after centrifugation at 5000 rpm for 2 min, followed by resuspension and fixation with 2.5% glutaraldehyde solution overnight (4 °C). The mixture was rinsed three times with 0.1 M PBS for 15 min, fixed with 1% osmic acid for 1 to 2 h and then rinsed three times with 0.1 M PBS. Then, the obtained sample was dehydrated by ethanol solutions with concentrations of 30%, 50%, 70%, 80%, 90%, and 95%, successively, and then treated as follows: 100% ethanol for 20 min, acetone solution for 20 min, acetone-embedding agent (V/V = 1/1) mixed solution for 1 h, acetone-embedding agent (V/V = 3/1) mixed solution for 3 h, and pure embedding agent for 12 h. After heating at 70 °C for 12 h, the embedded sample was sectioned with a Leica EM UC7 ultrathin microtome (70–90 nm), stained with lead citrate and 50% uranyl acetate ethanol solution for 5 min, and then observed with a transmission electron microscope.
4
Ultrastructural Analysis of Cauda Epididymis
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Fresh cauda epididymis tissue was removed and fixed overnight at 4 °C with 2.5% glutaraldehyde and 1% paraformaldehyde (diluted in PBS). The tissue was cut into 1-mm pieces and washed three times with 0.1 M PBS for 10 min each. The tissue was dehydrated with gradient ethanol and treated with pure propylene oxide for 15 min. Then, 25% Spurr’s resin, 50% Spurr’s resin, and 75% Spurr’s resin (prepared with propylene oxide) were used to replace the solution and incubated for 4 h each. Pure Spurr’s resin was used for replacement and incubated for more than 8 h before polymerization at 70 °C for 14 h. A Leica EM UC7 ultrathin microtome was used to cut the ultrathin sections at an 80 nm thickness using a DiATOME diamond knife. The ultrathin sections were cut on a single-hole carrier net covered with a Formvar membrane. The ultrathin sections were stained with 2% uranium dioxyacetate for 15 min and lead citrate for 10 min. Images were taken using a Hitachi HT7800 TEM.
5
Ultrastructural Analysis of the Blood-Brain Barrier
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The brains were removed and placed at 4°C on ice. Approximately 1-mm3 blocks of tissue from the prefrontal cortex were immediately placed in 2.5% glutaraldehyde solution, fixed at 4°C for 2∼4 h, and rinsed in 0.1 M phosphate buffer, pH 7.0, on a shaker 3 times for 15 min each. The tissues were placed in a 1% osmium acid solution for 1∼2 h and then rinsed with 0.1 M phosphate buffer, pH 7.0, on a shaker 3 times for 15 min each. The brain tissue blocks were dehydrated in gradient ethanol solutions (50, 70, 80, 90, and 95%; 15 min each), incubated with 100% ethanol for 20 min, and placed in pure acetone for 20 min. For gradient infiltration with an embedding agent, the tissues were incubated with acetone solution (V/V = 1/1) for 1 h and in acetone solution (V/V = 3/1) for 3 h and kept away from light at room temperature overnight. The tissue blocks were placed in the Leica EM UC 7 ultrathin microtome, ultrasectioned at a thickness of 70 nm. The tissues were stained with uranyl acetate 50% ethanol saturated solution for 15 min∼2 h and with lead citrate for 15 min. The structure of the BBB was observed by Transmission electron microscopy (TEM) (Hitachi H-7650).
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