For electron microscopy analysis, sorted cells were fixed in glutaraldehyde 2.5% (EMS) and osmium tetroxide 1% (EMS) overnight at 4 °C, followed by several washes with water and acetone (Sigma), and embedded in Epon (Sigma) resin the following day. Before imaging, 50 nm slides were prepared by using a Leica Ultracut microtome, and were contrasted using uranyl acetate (Sigma) and Reynolds lead citrate (Sigma). Electron microscope images were taken with a transmission electron microscope Philips CM100 at an acceleration of 80kV with a TVIPS TemCam-F416 digital camera with a magnification of 4800x and 11’000x. Image analysis and quantification were carried out using EMMENU, 3dmod (University of Colorado) and Fifi (ImageJ) software. To quantify mitochondria per sorted cell, a grid was applied and each intersection was defined as being part of the nucleus, cytoplasm or mitochondria and the length of each crista was measured divided by the mitochondrial area for determination of the crista density. For histology analysis, organs were trimmed and placed in the labeled cassettes and fixed in formalin for 24h for further embedding in molten paraffin wax. Paraffin sections at a thickness of 3-5 μm was stained with hematoxylin and eosin according to standard procedures. Images were taken and exported on a Nikon Eclipse Ti-S inverted microscope.
Ultracut microtome
Manufactured by Merck Group
The Ultracut microtome is a laboratory instrument used for precise sectioning of samples for microscopic analysis. It produces thin, uniform slices of material with a high degree of precision. The core function of the Ultracut microtome is to facilitate the preparation of samples for various imaging and analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti s inverted microscope, by Nikon (2 mentions)
Uranyl acetate, by Merck Group (2 mentions)
Cm100, by TVIPS (2 mentions)
Reynold s lead citrate, by Merck Group (2 mentions)
Temcam f416, by TVIPS (2 mentions)
Hematoxylin eosin he, by Merck Group (1 mentions)
Eg 1160 embedding center, by Leica (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
3 protocols using ultracut microtome
1
Comprehensive Electron Microscopy Analysis
2
Histological Processing and Imaging of Fish Heads
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Fish were sacrificed using 0.1 M MS-222 before the corneas were carefully opened to allow better penetration of the fixative. Subsequently, heads were excised and fixed in 4% paraformaldehyde in 0.1 M Phosphate buffer (pH 7.5) overnight at 4°C. For decalcification, heads were incubated in 0.5 M EDTA in 0.1 M Phosphate buffer for 3–4 days with daily exchange of the solution. Afterwards, heads were washed with ddH2O before being processed in a Paraffin-Infiltration-Processor (STP 420, Zeiss) according to the following protocol: ddH20: 1 min; 50% ethanol (EtOH) 5 min; 70% EtOH 10 min; 96% EtOH 25 min; 96% EtOH 2 × 20 min; 100% EtOH 2 × 20 min; xylene 2 × 20 min; paraffin 3 × 40 min/60°C; paraffin 60 min/60°C. The heads were embedded using the EG1160 Embedding Center (Leica). Semi-thin sections (2 µm) were cut using an Ultracut microtome and counterstained using hematoxylin/eosin (HE, Sigma). Paraffin embedding, sectioning and staining was performed by the CMCB Histology Facility. Imaging was performed using the ZEISS Axio Imager. Z1 provided by the CMCB Light Microscopy Facility and images were processed using Fiji. A total number of five fish per time point was analyzed.
3
Comprehensive Electron Microscopy Analysis
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For electron microscopy analysis, sorted cells were fixed in glutaraldehyde 2.5% (EMS) and osmium tetroxide 1% (EMS) overnight at 4 °C, followed by several washes with water and acetone (Sigma), and embedded in Epon (Sigma) resin the following day. Before imaging, 50 nm slides were prepared by using a Leica Ultracut microtome, and were contrasted using uranyl acetate (Sigma) and Reynolds lead citrate (Sigma). Electron microscope images were taken with a transmission electron microscope Philips CM100 at an acceleration of 80kV with a TVIPS TemCam-F416 digital camera with a magnification of 4800x and 11’000x. Image analysis and quantification were carried out using EMMENU, 3dmod (University of Colorado) and Fifi (ImageJ) software. To quantify mitochondria per sorted cell, a grid was applied and each intersection was defined as being part of the nucleus, cytoplasm or mitochondria and the length of each crista was measured divided by the mitochondrial area for determination of the crista density. For histology analysis, organs were trimmed and placed in the labeled cassettes and fixed in formalin for 24h for further embedding in molten paraffin wax. Paraffin sections at a thickness of 3-5 μm was stained with hematoxylin and eosin according to standard procedures. Images were taken and exported on a Nikon Eclipse Ti-S inverted microscope.
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