Tissues were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 (Science Services, Munich, Germany), postfixed in 2% aqueous osmium tetraoxide37 , dehydrated in gradual ethanol (30–100%) and propylene oxide, embedded in Epon (Merck, Darmstadt, Germany) and cured for 48 hours at 60 °C. Semithin sections were cut and stained with toluidine blue. Ultrathin sections of 50 nm were collected onto 200 mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (Zeiss Libra 120 Plus, Carl Zeiss NTS GmbH, Oberkochen, Germany). Pictures were acquired using a Slow Scan CCD-camera and iTEM software (Olympus Soft Imaging Solutions, Münster, Germany).
Slow scan ccd camera
Manufactured by Olympus
Sourced in Germany
The Slow Scan CCD-camera is a specialized imaging device designed for capturing high-quality images. It utilizes a charge-coupled device (CCD) sensor to convert light into digital signals, enabling the capture of detailed and low-noise images. The camera's core function is to provide a reliable and precise method for capturing visual data in various scientific and research applications.
Automatically generated - may contain errors
8 protocols using slow scan ccd camera
1
Electron Microscopy Tissue Preparation
2
Transmission Electron Microscopy Protocol
3
Electron Microscopy Tissue Sample Preparation
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Tissue samples were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 (Science Services, Munich, Germany), postfixed in 2% aqueous osmium tetraoxide (Dalton, 1955), dehydrated in gradual ethanol (30–100%) and propylene oxide, embedded in Epon (Merck, Darmstadt, Germany) and cured for 24 hours at 60°C. Semi-thin sections were cut and stained with toluidine blue. Ultrathin sections of 50 nm were collected onto 200 mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (Zeiss Libra 120 Plus, Carl Zeiss NTS GmbH, Oberkochen, Germany). Pictures were acquired using a Slow Scan CCD-camera and iTEM software (Olympus Soft Imaging Solutions, Muenster, Germany).
4
Ultrastructural Analysis of Autophagic Bodies
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After treatment, cells were collected into a 1.5 mL EP tube, and a low‐speed centrifugation step was employed to settle the cells at the bottom of the EP tube. Subsequently, the supernatants were removed. The collected cell pellets were fixed with 2.5% glutaraldehyde for 4 h at 4 °C and post‐fixed in 1% osmium tetroxide for 2 h at 20 °C. Then, they were dehydrated using a gradient of ethanol (50–100%) and acetone, embedded in epoxy resin, and polymerized for 48 h at 60 °C. Ultrathin sections (80 nm) were cut and stained with uranyl acetate and lead citrate prior to transmission electron microscopy (HT7700, HITACHI). Images were captured using a SlowScan CCD camera and the iTEM software (Ver 01.07, Olympus Soft Imaging Solutions). The quantification of autophagic bodies followed established procedures as previously described.[48 (link)
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Analyzing Mitochondrial Morphology and Function
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Mito-Tracker Red or Blue dye was added to each cell dish at a final concentration of 50 nM. The cells were incubated with the dye for 15–20 min at 37 °C. After incubation, cells were fixed with 4% PFA for 1 h and then washed three times with PBS. Images were obtained using a Nikon Eclipse E600 microscope (Nikon, Melville, NY). Mitochondrial area and form (length and circularity) were analyzed by ImageJ. TEM was used to analyze mitochondrial morphology. Cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4, postfixed in 2% aqueous osmium tetroxide, dehydrated in gradual ethanol (30–100%) and propylene oxide, embedded in Epon (Merck, Darmstadt, Germany) for 24 h at 60 °C. Ultra-thin sections were cut and stained with 0.5% uranyl acetate and 3% lead citrate at 20 °C for 30 and 7 min, respectively before TEM analysis (Zeiss Libra 120 Plus, Carl Zeiss NTS GmbH, Oberkochen, Germany). Pictures were taken using a Slow Scan CCD-camera and iTEM software (Olympus Soft Imaging Solutions, Münster, Germany).
6
Ultrastructural Analysis of Sms1 Knockout Testes
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Animals (Sms1-/- n = 2, SMS1+/+ n = 2, age: 12 weeks) were transcardially perfused with 50ml ice cold PBS, prior to perfusion with 70ml of fixing solution (2.5% PFA, 2.5% glutaraldehyde in PBS). Testes and epididymides were removed and cut into 1mm3 cubes, which were post fixed in 2.5% glutaraldehyde containing 0.1M sodium cacodylate buffer (pH 7.4) at 4°C over night.
Tissues were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 (Science Services, Munich, Germany), postfixed in 2% aqueous osmium tetraoxide (Dalton, 1955), dehydrated in gradual ethanol (30–100%) and propylene oxide, embedded in Epon (Merck, Darmstadt, Germany) and cured for 24 hours at 60°C. Semithin sections were cut and stained with toluidine blue. Ultrathin sections of 50 nm were collected onto 200 mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (Zeiss Libra 120 Plus, Carl Zeiss NTS GmbH, Oberkochen, Germany). Pictures were acquired using a Slow Scan CCD-camera and iTEM software (Olympus Soft Imaging Solutions, Münster, Germany).
Tissues were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 (Science Services, Munich, Germany), postfixed in 2% aqueous osmium tetraoxide (Dalton, 1955), dehydrated in gradual ethanol (30–100%) and propylene oxide, embedded in Epon (Merck, Darmstadt, Germany) and cured for 24 hours at 60°C. Semithin sections were cut and stained with toluidine blue. Ultrathin sections of 50 nm were collected onto 200 mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (Zeiss Libra 120 Plus, Carl Zeiss NTS GmbH, Oberkochen, Germany). Pictures were acquired using a Slow Scan CCD-camera and iTEM software (Olympus Soft Imaging Solutions, Münster, Germany).
7
Ultrastructural Analysis of Mitochondria in Frozen Tissues
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For transmission electron microscopy (TEM) analysis, fresh‐frozen tissues were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, and TEM fixation buffer (Electron Microscopy Sciences, Hatfield, USA) at 4 °C. The samples were subsequently post‐fixed in 2% aqueous osmium tetraoxide 44, dehydrated in increasing concentrations of ethanol (30–100%) and propylene oxide, embedded in Epon (Merck, Darmstadt, Germany), and dried for 24 h at 60 °C. Semithin sections were cut and stained with toluidine blue. Ultrathin sections of 50 nm were collected on 200‐mesh copper grids and stained with uranyl acetate and lead citrate before examination by TEM (Zeiss Libra 120 Plus, Carl Zeiss NTS GmbH, Oberkochen, Germany). Images were acquired using a Slow Scan CCD camera and iTEM software (Olympus Soft Imaging Solutions, Münster, Germany). The number of mitochondria was determined by manually counting the mitochondria per field of view in longitudinally sectioned fibres, viewed at 1600× magnification.
8
Transmission Electron Microscopy Analysis
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For TEM analysis, about 105 labeled or infected cells were washed with PBS after trypsinization. Free MNP suspensions, free MNP-VP complexes, both containing about the same amount of iron as that applied to cells, and the cells were pelleted for 5 minutes at 1,600 rpm in beam tubes and the (cell) pellets were fixed with 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 TEM fixation buffer (Electron Microscopy Sciences, Hatfield, United States) at 4 °C. The samples were then post-fixed in 2 % aqueous osmium tetraoxide 44 , dehydrated in gradual ethanol (30 - 100 %) and propylene oxide, embedded in Epon (Merck, Darmstadt, Germany) and dried for 24 hours at 60 °C. Semithin sections were cut and stained with toluidine blue. Ultrathin sections of 50 nm were collected onto 200 mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (Zeiss Libra 120 Plus, Carl Zeiss NTS GmbH, Oberkochen, Germany). Pictures were acquired using a Slow Scan CCD-camera and iTEM software (Olympus Soft Imaging Solutions, Münster, Germany).
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