The samples assayed by the co-sedimentation were also analysed by electron microscopy. Copper grids coated with formvar and carbon were prepared with Leica EM ACE200 carbon coating system. Grids were glow-discharged with Pelco easiGlow system (Ted Pella, Inc., Redding, CA, USA). 3.5 µl sample adsorbed for 2 min, washed twice in water and immediately stained in 50 µl 1.5% uranyl acetate solution for 30 s. Samples were examined with Talos 120C (FEI, Eindhoven, The Netherlands) operating at 120 kV. Micrographs were acquired with a Ceta 16M CCD camera (FEI, Eindhoven, The Netherlands) using TEM Image & Analysis software ver. 4.15 (FEI, Eindhoven, The Netherlands).
Tem image analysis software ver 4
Manufactured by Thermo Fisher Scientific
TEM Image & Analysis software ver. 4.15 is a software application designed for the analysis and processing of transmission electron microscopy (TEM) images. The software provides tools for image capture, enhancement, and quantitative analysis of TEM data.
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Lab products found in correlation
Ceta 16m ccd camera, by Thermo Fisher Scientific (4 mentions)
Talos 120c, by Thermo Fisher Scientific (2 mentions)
Pelco easiglow system, by Ted Pella (1 mentions)
Em ace200 carbon coating system, by Leica (1 mentions)
Tem 1230 microscope, by JEOL (1 mentions)
Glutaraldehyde, by TAAB (1 mentions)
Osmium tetroxide, by TAAB (1 mentions)
Paraformaldehyde (pfa), by TAAB (1 mentions)
Spurr resin, by TAAB (1 mentions)
4 protocols using tem image analysis software ver 4
1
Electron Microscopy of Protein Samples
2
Negative Staining Electron Microscopy Protocol
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Negative staining EM: Samples were applied to carbon-coated copper-grids and stained with 1.5% Uranyl Acetate (UA). The 41PB sample was analysed with a JEOL-1230 TEM-Microscope (JEOL, Japan) at 80 kV and micrographs were acquired with a Orius 830 2k × 2k CCD camera using the digital micrograph software (Gatan, US) and a pixel size of 2.69 Å on the objective scale. The 5PB sample was analysed with a Talos 120 C TEM-Microscope (FEI, The Netherlands) operating at 120 kV and micrographs were acquired with a Ceta 16 M CCD camera (FEI) using the TEM Image & Analysis software ver. 4.15 (FEI) and a pixel size of 1.56 Å on the objective scale.
3
Pine Needle Chloroplast Ultrastructure Analysis
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Thin slices (< 0.5 mm) from the middle region of pine needles were cut in tap water and fixed in 4% paraformaldehyde, 2.5% glutaraldehyde (TAAB Laboratories, Aldermaston, England) in 0.1 M or 0.05 M (May and June) sodium cacodylate buffer, pH 7.4 (TAAB Laboratories, Aldermaston, England). Thoroughly washed samples were post-fixed in 1% osmium tetroxide (TAAB Laboratories, Aldermaston, England). The fixed material was dehydrated in ethanol series with increasing concentrations and propylene oxide and finally embedded in Spurr resin (TAAB Laboratories, Aldermaston, England). Ultrathin sections (70 nm) were post contrasted in uranyl acetate and Reynolds lead citrate and further examined with Talos 120 C electron microscope (FEI, Eindhoven, The Netherlands) operating at 120 kV. Micrographs were acquired with a Ceta 16 M CCD camera (FEI, Eindhoven, The Netherlands) using TEM Image & Analysis software ver. 4.14 (FEI, Eindhoven, The Netherlands). The chloroplast ultrastructure was analyzed from the electron micrographs by measuring the average number of chloroplasts per cell, average number of grana per chloroplasts and average number of appressed thylakoids per grana stack (Ng)65 ,66 (link).
4
Ultrastructural Examination of Peri-Implant and Periodontal Tissues
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Two patients participated in the present study, one with a severe peri‐implantitis and one with a severe periodontitis. Both patients were scheduled for surgical periodontal treatment at the specialist clinic in periodontology, University Hospital of Umeå, Sweden. Informed written approval was given by both subjects, and authorization for the study was obtained from the Regional Ethical Review Board at Umea University, Sweden (Dnr. 2013–337‐31M, 2016–417‐32M). Tissue removed during surgery from the peri‐implant and periodontal mucosa, respectively, was obtained for examination with transmission electron microscopy. The peri‐implantitis patient had a prosthetic supraconstruction made of Ti‐porcelain without an abutment and the periodontitis patient periodontal surgery was performed on a tooth reconstructed with amalgam and composite.
Tissues were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, cut into smaller pieces and post fixed in 1% osmium tetroxide, dehydrated with ethanol, a final step in propylene oxide and embedded in resin according to standard procedures. Sections were contrasted with uranyl acetate, Reynold's lead citrate and examined with a Talos 120 C (FEI) operating at 120 kV. Micrographs were acquired with a Ceta 16 M CCD camera (FEI) using TEM Image & Analysis software ver. 4.14 (FEI)
Tissues were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, cut into smaller pieces and post fixed in 1% osmium tetroxide, dehydrated with ethanol, a final step in propylene oxide and embedded in resin according to standard procedures. Sections were contrasted with uranyl acetate, Reynold's lead citrate and examined with a Talos 120 C (FEI) operating at 120 kV. Micrographs were acquired with a Ceta 16 M CCD camera (FEI) using TEM Image & Analysis software ver. 4.14 (FEI)
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