Biopsies (2 mm sections) were taken out from four different paraffin blocks containing samples of native, detergent treated, pressurized CO2-EtOH-H2O treated or pressurized CO2-limonene treated. The samples had previously been sectioned for H&E staining and all had been treated with benzonase nuclease, except for the native control. The biopsy was dewaxed in xylene, washed in ethanol, stained with 0.05% methylene blue in ethanol, rinsed in ethanol, acetone, followed by 1:1 mixture of Polybed-acetone and finally embedded in pure Polybed 812. The polymerised block was sectioned with a Leica UC7 ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany) and sections were mounted on a pioloform coated copper Maxtaform H5 grid. The section was contrasted with 4% Uranyl acetate followed with 1% lead citrate. Images of the samples were analysed in a Tecnai BioTWIN transmission electron microscope (FEI Company, OR, USA) at two different magnifications.
Tecnai bio twin transmission electron microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Tecnai Bio Twin transmission electron microscope is a high-performance instrument designed for biological and material science applications. It provides high-resolution imaging capabilities for studying the structure and morphology of various samples at the nanoscale level.
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Lab products found in correlation
10nm gold particles, by Aurion (5 mentions)
Amt ccd camera, by Advanced Microscopy Techniques (4 mentions)
Monoclonal anti cd9, by Abcam (2 mentions)
Ultracut uc7, by Leica camera (2 mentions)
Embed 812, by Electron Microscopy Sciences (2 mentions)
Morada ccd camera, by Olympus (2 mentions)
Leica uc7 ultramicrotome, by Leica Microsystems (1 mentions)
Anti 53bp1, by Fortis Life Sciences (1 mentions)
Goat anti rabbit igg biotinylated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Vectastain abc hrp kit, by Vector Laboratories (1 mentions)
Anti connexin 43, by Merck Group (1 mentions)
Item software, by Olympus (1 mentions)
Qnano analysis, by Izon Science (1 mentions)
Anti ibsp, by Thermo Fisher Scientific (1 mentions)
Anti cd63, by Santa Cruz Biotechnology (1 mentions)
Eclipse 80i light microscope, by Nikon (1 mentions)
Rm2135 microtome, by Leica Biosystems (1 mentions)
Histostar embedding center, by Thermo Fisher Scientific (1 mentions)
Ab92726, by Abcam (1 mentions)
17 protocols using tecnai bio twin transmission electron microscope
1
Ultrastructural Analysis of CO2-based Biomass Treatments
2
Immunogold Labeling of DNA Damage Proteins
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Tissue samples were diced into small cubes of 2 mm3 and fixed overnight with paraformaldehyde and glutaraldehyde. Fixed tissue samples were dehydrated in increasing concentrations of alcohol and infiltrated with LR Gold resin (EMS, Hatfield, PA). Finally, samples were embedded in fresh resin with benzil (EMS) and polymerizated with ultraviolet light illumination. Ultrathin sections were cut on an Ultracut UCT Leica with diamond knives (Diatome; Biel, Switzerland), picked up with pioloform-coated nickel grids, and processed for immuno-labeling. To block unspecific staining, sections were floated on glycine followed by blocking solution (EMS). Afterwards, sections were incubated with the primary antibody (anti-53BP1, Bethyl Laboratories; anti-pKu70 (pSer6), AbcamInc, Cambridge, MA, USA). After rinsing, secondary antibody conjugated with 6-nm or 10-nm goldparticles (EMS) was applied to the grids. Subsequently, sections were rinsed and post-fixed with glutaraldehyde. Immunogold-labeling omitting the primary antibody was used as control. All sections were stained with uranyl acetate and examined using a TecnaiBiotwin transmission electron microscope (FEI Company, Eindhoven, The Netherlands).
3
Immunogold Labeling of PD-L1 in TEM
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Fixed specimens at an optimal concentration were placed onto a 400 mesh carbon/formvar coated grids and allowed to absorb to the formvar for a minimum of 1 min. The grids were placed into a blocking buffer for a block/permeabilization step for one hour. Without rinsing, the grids were immediately placed into the primary antibody at the appropriate dilution overnight at 4 °C (Mouse anti-PD-L1, sc-293425, Santa Cruz). As controls, some of the grids were not exposed to the primary antibody. The following day, all the grids were rinsed with PBS then floated on drops of the appropriate secondary antibody attached with 10 nm gold particles (AURION, Hatfield, PA) for two hours at room temperature. Grids were rinsed with PBS and were placed in 2.5% Glutaraldehyde in 0.1 M Phosphate buffer for 15 min. After rinsing in PBS and distilled water, the grids were allowed to dry and stained for contrast using uranyl acetate. The samples were viewed with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, OR) and images were taken with an AMT CCD Camera (Advanced Microscopy Techniques, Danvers, MA).
4
Ultrastructural Examination of Neuronal Cell Death
5
Cerebral Organoids Ultrastructural Analysis
6
Negative Staining of Concentrated tecVLP Samples
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Concentrated tecVLP-containing supernatants were fixed by incubation with an equal volume of 5% paraformaldehyde and stored at 4°C until use. The concentrated, fixed tecVLP samples were processed as follows. First, 2 μL of each sample was pipetted onto a 300-mesh formvar/carbon-coated nickel grid (EMS, Hatfield, PA, USA), and the sample was incubated overnight at 4°C. The samples were then blotted, rinsed with bacitracin (50 μg/mL) [23 (link)], blotted, negatively stained with 5% ammonium molybdate (pH 6.9) and 0.1% (w/v) trehalose, and blotted a final time [24 ]. The grid was examined using a Tecnai BioTwin transmission electron microscope (FEI Company, Hillsboro, OR, USA) operating at 120 kV, and images were captured with a 2K × 2K camera (AMT Corp., Woburn, MA, USA).
7
Immunogold Labeling for Electron Microscopy
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Fixed specimens at an optimal concentration were placed onto a 300 mesh
carbon/formvar coated grids and allowed to absorb to the formvar for a minimum
of 1 minute. For immunogold staining the grids were placed into a blocking
buffer for a block/permeabilization step for 1 hour. Without rinsing, the grids
were immediately placed into the primary antibody at the appropriate dilution
overnight at 4°C (monoclonal anti-CD9 1:10, Abcam). As controls, some
grids were not exposed to the primary antibody. The next day, all of the grids
were rinsed with PBS then floated on drops of the appropriate secondary antibody
attached with 10nm gold particles (AURION, Hatfield, PA) for 2 hours at room
temperature. Grids were rinsed with PBS and were placed in 2.5%
Glutaraldehyde in 0.1M phosphate buffer for 15 minutes. After rinsing in PBS and
distilled water the grids were allowed to dry and stained for contrast using
uranyl acetate. The samples were viewed with a Tecnai Bio Twin transmission
electron microscope (FEI, Hillsboro, OR) and images were taken with an AMT CCD
Camera (Advanced Microscopy Techniques, Danvers, MA).
carbon/formvar coated grids and allowed to absorb to the formvar for a minimum
of 1 minute. For immunogold staining the grids were placed into a blocking
buffer for a block/permeabilization step for 1 hour. Without rinsing, the grids
were immediately placed into the primary antibody at the appropriate dilution
overnight at 4°C (monoclonal anti-CD9 1:10, Abcam). As controls, some
grids were not exposed to the primary antibody. The next day, all of the grids
were rinsed with PBS then floated on drops of the appropriate secondary antibody
attached with 10nm gold particles (AURION, Hatfield, PA) for 2 hours at room
temperature. Grids were rinsed with PBS and were placed in 2.5%
Glutaraldehyde in 0.1M phosphate buffer for 15 minutes. After rinsing in PBS and
distilled water the grids were allowed to dry and stained for contrast using
uranyl acetate. The samples were viewed with a Tecnai Bio Twin transmission
electron microscope (FEI, Hillsboro, OR) and images were taken with an AMT CCD
Camera (Advanced Microscopy Techniques, Danvers, MA).
8
Ultrastructural Analysis of Mitochondria
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Briefly, the brain tissue sections were postfixed with 4% paraformaldehyde +2.5% glutaraldehyde in 0.1 M PB for 14 hours at 4°C. Then, they were washed and stored in 0.1 M PB [34 (link)]. The coverslips were processed for electron microscopy as previously described with modifications [35 (link)]. Ultrathin sections at 70-80 nm were cut on an ultramicrotome and collected on Formvar-coated single slot grids (Electron Microscopy Sciences). Grids were stained with uranyl acetate and lead citrate solutions, dried, and stored in a grid box for EM imaging. Micrographs were taken on a Tecnai Biotwin transmission electron microscope (FEI, Hillsboro, Oregon, USA). We measured mitochondrial size as the percentage area of the total area of the cytoplasm [36 (link)] (comprising >1800 mitochondria in total) using ImageJ v.1.49 (http://imagej.nih.gov/ij/ ). Analysis was performed by an investigator blinded to treatment group assignment.
9
Transmission Electron Microscopy of Tissues
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TEM was performed by the Yale Biological Electron Microscopy facility. To process tissues for TEM, adipose, pancreatic, and kidney tissue was isolated from mice and immediately cut into small pieces (1 µm × 1 µm × 1 µm) in ice-cold PBS. The tissue pieces were gently shaken with electron microscopy fixation buffer (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate [pH 7.4]) at room temperature for 30 min and then incubated at 4°C for another 90 min. The tissue samples were washed three times with 0.1 M Na cacodylate buffer (pH 7.4).
To process cells for TEM, differentiated adipocytes (day 8) in 10-cm culture dishes were fixed in prewarmed 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) at room temperature for 30 min and then incubated at 4°C for another 60 min. The cells were washed three times with 0.1 M sodium cacodylate buffer (pH 7.4).
Subsequent processing was performed by the Yale electron microscopy facility. Specimens were viewed on a FEI Tecnai BioTWIN transmission electron microscope at an accelerating voltage of 80 kV, and images were obtained with a SIS Morada 11-megapixel charge-coupled device camera and iTEM software (Olympus).
To process cells for TEM, differentiated adipocytes (day 8) in 10-cm culture dishes were fixed in prewarmed 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) at room temperature for 30 min and then incubated at 4°C for another 60 min. The cells were washed three times with 0.1 M sodium cacodylate buffer (pH 7.4).
Subsequent processing was performed by the Yale electron microscopy facility. Specimens were viewed on a FEI Tecnai BioTWIN transmission electron microscope at an accelerating voltage of 80 kV, and images were obtained with a SIS Morada 11-megapixel charge-coupled device camera and iTEM software (Olympus).
10
Characterization of Extracellular Vesicles
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Samples were absorbed onto 200 mesh glow-discharged carbon-coated grids for 2 min and subsequently dried at room temperature. Then, samples were stained with 2% (w/v) uranyl acetate (UrAc) on ice for 5 min and blotted-off for excess UrAc. Subsequently, the grids were observed under a FEI Tecnai BioTwin Transmission Electron Microscope (FEI Company, OR, USA). The qNano analysis (Izon Science, USA) was used to characterize extracellular vesicles diluted in PBS according to the manufacturer’s instructions.
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