Tumor-implanted animals were perfused with PBS followed by a fixation solution of 2.5% glutaraldehyde, 4% PFA in 0.1 M Sodium Cacodylate (Caco) buffer, pH 7.4. After fixation, the brain was removed and sectioned into 100 - 500 μM slices. Under a fluorescent dissection microscope, tissue punches were taken to capture tumor and non-tumor areas. Punches were placed in fixation solution overnight at 4°C. Samples were washed 2 times for 15 min in 0.1M Caco buffer, pH 7.4 and then immersed in 1% osmium diluted in 0.1M Caco buffer, pH 7.4 for 50 min at room temperature on a rotator in the dark. Samples were then washed 4 times in 0.2M Caco buffer, pH 7.4. Samples were dehydrated in 50% 80% and 95% acetone for 5 min each. 4 washes in 100% acetone for 15 min were performed. Samples were equilibrated for embedding in a solution containing equal parts of 100% acetone and Epon 812 with accelerator on a rotator overnight at room temperature. Samples were embedded in 100% Epon 812 with the accelerator at 60-70°C overnight. Ultrathin sections were cut and imaged on a Tecnai T12 120kV Transmission Electron Microscope (FEI).
Tecnai t12 120kv transmission electron microscope
Manufactured by Thermo Fisher Scientific
The Tecnai T12 120kV Transmission Electron Microscope is a high-performance electron microscope designed for advanced imaging and analysis of samples. It operates at a maximum accelerating voltage of 120 kilovolts and is capable of providing high-resolution, high-contrast images of a wide range of materials and specimens.
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2 protocols using tecnai t12 120kv transmission electron microscope
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Fixation and Embedding for TEM Imaging
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Fixation and Embedding for TEM Imaging
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Tumor-implanted animals were perfused with PBS followed by a fixation solution of 2.5% glutaraldehyde, 4% PFA in 0.1 M Sodium Cacodylate (Caco) buffer, pH 7.4. After fixation, the brain was removed and sectioned into 100 - 500 μM slices. Under a fluorescent dissection microscope, tissue punches were taken to capture tumor and non-tumor areas. Punches were placed in fixation solution overnight at 4°C. Samples were washed 2 times for 15 min in 0.1M Caco buffer, pH 7.4 and then immersed in 1% osmium diluted in 0.1M Caco buffer, pH 7.4 for 50 min at room temperature on a rotator in the dark. Samples were then washed 4 times in 0.2M Caco buffer, pH 7.4. Samples were dehydrated in 50% 80% and 95% acetone for 5 min each. 4 washes in 100% acetone for 15 min were performed. Samples were equilibrated for embedding in a solution containing equal parts of 100% acetone and Epon 812 with accelerator on a rotator overnight at room temperature. Samples were embedded in 100% Epon 812 with the accelerator at 60-70°C overnight. Ultrathin sections were cut and imaged on a Tecnai T12 120kV Transmission Electron Microscope (FEI).
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