Fixed retinae were washed in 0.1 M PB (3 × 10 min), dehydrated in a series of 50 to 100% acetone and incubated in a 1:1 mixture of acetone and Agar 100 Resin (Agar Scientific) for 1 h at RT. After incubation in pure Agar 100 Resin overnight at RT, embedding medium was hardened for 48 h at 60°C. Embedded retinae were cut using a Reichert-Jung Ultracut E ultramicrotome. Semithin sections (0.5 μm thickness) were collected on slides, stained with 2% toluidine blue and 0.5% sodium borate in double-distilled water and examined with a Leica DM6 microscope. Ultrathin sections (90 nm thickness) were collected on copper grids and analyzed using a Zeiss EM 902A electron microscope. Electron micrographs were adjusted in brightness and contrast using Adobe Photoshop CS6 Extended (Adobe Systems).
Em902a electron microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss EM902A is a transmission electron microscope designed for high-resolution imaging and analysis of materials at the nanoscale. It features a lanthanum hexaboride (LaB6) electron source, which provides high brightness and stability, enabling detailed observation of fine structural details. The EM902A is capable of achieving a resolution of up to 0.5 nanometers, allowing users to examine the atomic-level structure of a wide range of specimens.
Automatically generated - may contain errors
Lab products found in correlation
Fastscan ccd camera 1 024 x 1 024, by TVIPS (2 mentions)
Agar 100 resin, by Agar Scientific (1 mentions)
Ultracut e ultramicrotome, by Reichert Technologies (1 mentions)
Em uc7 ultra microtome, by Zeiss (1 mentions)
Emmanu4 v, by TVIPS (1 mentions)
Fastscan ccd camera, by TVIPS (1 mentions)
Formvar carbon coated 100 mesh grids, by Quantifoil (1 mentions)
Ultracut s, by Reichert Technologies (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Carbon coated copper grids, by Quantifoil (1 mentions)
11 protocols using em902a electron microscope
1
Transmission Electron Microscopy of Retina
2
Microscopic Analysis of Scutigera Tracheal System
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Specimens of Scutigera coleoptrata Drosophila Musca
For scanning electron microscopic (SEM) investigations of the tracheal tubules, whole specimens were fixed in ethanol (70%). Tracheae were macerated using pepsin. Optimal results were obtained with a solution of 1–2 g pepsin in 100 ml HCl (37%, Hilken 1994 , 1998 ). The complex of tracheal tubules was dehydrated through a graded series of ethanol, critical-point dried, coated with gold, and studied with a CAMSCAN DV4.
For light microscopic (LM) and transmission electron microscopic (TEM) investigations, segments ofScutigera coleoptrata
For scanning electron microscopic (SEM) investigations of the tracheal tubules, whole specimens were fixed in ethanol (70%). Tracheae were macerated using pepsin. Optimal results were obtained with a solution of 1–2 g pepsin in 100 ml HCl (37%, Hilken 1994 , 1998 ). The complex of tracheal tubules was dehydrated through a graded series of ethanol, critical-point dried, coated with gold, and studied with a CAMSCAN DV4.
For light microscopic (LM) and transmission electron microscopic (TEM) investigations, segments of
3
Quantifying Autophagosomes in VacA-Treated AGS Cells
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AGS cells were treated with 50 ng/ml VacAs1m1 toxin for 6 h, collected, and fixed in a glutaraldehyde and sodium cacodylate solution for 2 h, and then fixed with 1% OsO4 for 1 h and a half, and then stained in 3% aqueous uranyl acetate for 1 h. These cells were dehydrated with graded alcohol (50%, 70%, 80%, 90%, 95%, 100%) and embedded in Epon-Araldite resin (Canemco, #034). Ultrathin sections were prepared on a Leica EM UC7 ultramicrotome, counterstained with 0.3% lead citrate, and observed under a Zeiss EM 902A electron microscope. The autophagosome counting method was followed as described previously by Yla-Anttila et al31 (link).
4
Freeze-Fracture Replica Imaging of Virus Vesicles
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Vesicles isolated from 5 × 104 infected monocytes (MOI 1:1) were collected in 5-µL suspension and enclosed between two 0.1-mm-thick copper profiles as used for the freeze-fracture sandwich double-replica technique. The sandwiches were physically fixed by rapid plunge-freezing in a liquid ethane/propane mixture, cooled by liquid nitrogen. Freeze-fracturing was performed at −150 °C in a BAF400T freeze-fracture unit (BAL-TEC) using a double-replica stage. The fractured samples were shadowed with 2 nm Pt/C (platinum/carbon) at an angle of 35 °C, followed by perpendicular evaporation of a 15–20-nm-thick carbon layer. The evaporation of Pt/C was controlled by a thin-layer quartz crystal monitor; the thickness of the carbon layer was controlled optically. The obtained freeze-fracture replica was transferred to a cleaning solution (commercial sodium hypochlorite, containing 12% active Cl2) for 30 min at 45 °C. Then, the replica was washed four times in distilled water and transferred onto unfiled copper EM-grids for examination in a Zeiss EM902A electron microscope (Carl Zeiss) operated at 80 kV. Images were recorded with a 1 k (1024 × 1024) FastScan-CCD-camera (CCD-camera and acquisition software EMMANU4 v 4.00.9.17, TVIPS).
5
Visualizing Emp Protein Structures
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Carbon-coated grids (400 meshes) (Quantifoil, Großlöbichau, Germany) were hydrophilized by glow discharge at low pressure in air. Subsequently, 20 μl a solution of purified EmpFL (3.1nmol) or Emp2+3 (2.4nmol), was adsorbed onto the hydrophilic grids for 1 min. The grids were washed twice with drops of distilled water and were stained with a drop of 2% uranyl acetate in distilled water. The samples were analysed using a Zeiss EM902A electron microscope (Carl Zeiss AG, Oberkochen, Germany) operated at an acceleration voltage of 80 kV. Images were recorded with a FastScan-CCD camera at 1024 × 1024 pixels (TVIPS, Munich, Germany).
6
Visualizing C1q-ApoE Complexes by TEM
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To visualize single C1q protein particles by TEM, C1q (5 μg/ml)
was diluted in PBS. In order to gold-label ApoE, biotinylated plasma-purified
ApoE or ApoE139-152 (20 μg/ml) was incubated with
streptavidin-gold complexes (5 nm gold, British BioCell International Ltd.)
diluted 1:25 in PBS for two hours at RT. C1q (10 μg/ml) was added to the
ApoE-streptavidin-gold solution (1:1 mixture) and incubated under gentle shaking
for two hours at RT. To detect single C1q-ApoE3 complexes by structure, full
length ApoE3 (40 μg/ml) was directly labeled with EM-Grade 6 nm gold
particles (AURION-ImmunoGold Reagents & Accessories, The Netherlands).
The probe (containing ~2 x 1014 gold particles/ml) was diluted
1:200 in PBS. Carbon-coated grids were hydrophilized by glow discharge at low
pressure in air. Aliquots of C1q alone and C1q-ApoE-streptavidin-gold or
C1q-ApoE3-gold were adsorbed onto hydrophilic, carbon-coated grids for 1 min,
washed twice with ddH2O, and stained on a drop of 2% uranyl acetate
in ddH2O. Specimens were analyzed with a Zeiss EM902A electron
microscope (Carl Zeiss) operated at 80 kV accelerating voltage, and images were
recorded with a FastScan-CCD-camera 1,024 x 1,024 (TVIPS).
was diluted in PBS. In order to gold-label ApoE, biotinylated plasma-purified
ApoE or ApoE139-152 (20 μg/ml) was incubated with
streptavidin-gold complexes (5 nm gold, British BioCell International Ltd.)
diluted 1:25 in PBS for two hours at RT. C1q (10 μg/ml) was added to the
ApoE-streptavidin-gold solution (1:1 mixture) and incubated under gentle shaking
for two hours at RT. To detect single C1q-ApoE3 complexes by structure, full
length ApoE3 (40 μg/ml) was directly labeled with EM-Grade 6 nm gold
particles (AURION-ImmunoGold Reagents & Accessories, The Netherlands).
The probe (containing ~2 x 1014 gold particles/ml) was diluted
1:200 in PBS. Carbon-coated grids were hydrophilized by glow discharge at low
pressure in air. Aliquots of C1q alone and C1q-ApoE-streptavidin-gold or
C1q-ApoE3-gold were adsorbed onto hydrophilic, carbon-coated grids for 1 min,
washed twice with ddH2O, and stained on a drop of 2% uranyl acetate
in ddH2O. Specimens were analyzed with a Zeiss EM902A electron
microscope (Carl Zeiss) operated at 80 kV accelerating voltage, and images were
recorded with a FastScan-CCD-camera 1,024 x 1,024 (TVIPS).
7
Cryo-TEM Imaging of Outer Membrane Vesicles
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A drop of a freshly prepared OMV solution (2 μl) was placed on a R3.5/1 holey carbon-coated copper grid (Quantifoil Micro Tools GmbH, Germany). The sample was rapidly plunge-frozen in liquid ethane at −180°C. The frozen grid was transferred into a liquid nitrogen-cooled Gatan 626-DH cryo-holder (Gatan Inc., USA) and inserted into a Philips CM 120 cryo-TEM (Philips, Netherlands) operated at a 120-kV accelerating voltage. Images were acquired with a 1k × 1k FastScan-F114 charge-coupled device (CCD) camera (TVIPS GmbH, Germany). An isolated OMV/minicell solution (20 μl) was placed for 1 min onto hydrophilic, Formvar/carbon-coated copper grids (Quantifoil Micro Tools GmbH, Germany), washed twice on drops of distilled water, and stained on a drop of 2% uranyl acetate in distilled water. Samples were imaged in a Zeiss EM902A electron microscope (Carl Zeiss AG, Germany) operated at an 80-kV accelerating voltage. Images were acquired with a 1k × 1k FastScan-F114 CCD camera (TVIPS GmbH, Germany).
8
Visualizing C1q-ApoE Complexes by TEM
9
Tissue Fixation and Ultrathin Sectioning for Electron Microscopy
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After hyperthermia treatment and euthanasia, the residual tumor tissues were excised. Pieces of tissue of 1 mm size were fixed for 3 h at room temperature and then overnight at 4 °C with 4% (v/v) formaldehyde (freshly prepared from paraformaldehyde) and 2.5% (v/v) glutaraldehyde in sodium cacodylate buffer (0.1 M and pH 7.2). The tissue samples were washed 3 times for about 30 min with sodium cacodylate buffer and postfixed with 1% (w/v) osmium tetroxide in cacodylate buffer for 2 h at 20 °C. Samples were dehydrated in an ascending ethanol series and stained with 2% (w/v) uranyl acetate in 50% (v/v) ethanol. The samples were embedded in Araldite resin (Plano, Wetzlar, Germany) according to manufacturer’s instruction. Ultrathin sections of 70 nm thickness were cut using an ultramicrotome Ultracut S (Reichert-Jung, Vienna, Austria) and mounted on Formvar-carbon-coated 100 mesh grids (Quantifoil, Großlöbichau, Germany). The Ultrathin sections were stained with lead citrate for 10 min and examined in a Zeiss EM 902A electron microscope (Carl Zeiss AG, Oberkochen, Germany) operated at 80 kV. Digitized images were taken with a Wide-angle Dual Speed 2K CCD camera controlled by a Sharp:Eye base controller and operated by the Image SP software (camera and software: TRS, Moorenweis, Germany).
10
TEM Imaging of Organ Tissues
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For transmission electron microscopy, organ tissues were fixed in 5% glutaraldehyde in 0.4 M PBS buffer (pH 7.2–7.3). The samples were washed three times with 0.1 M cacodylate buffer and stored in this buffer at 4°C until plastic embedding. Afterwards preparations were postfixed with 2% osmium tetraoxide in 0.1 M cacodylate buffer for 2 hours at 4°C. Before embedding in araldite (Novartis Pharma, Nürnberg, Germany) the specimens were dehydrated in a graded series of ethanol. Sections of plastic-embedded specimens were cut with a diamond knife for thin sections and ultrathin sections on an ultra-microtome (Reichert, Buffalo, NY, USA). The 0.5-μm thin slices were stained with Methylene Blue and examined using a Zeiss Axiovert 200 (Zeiss, Oberkochen, Germany). Ultrathin sections (60 nm) were mounted on formvar-coated copper grids, stained with 0.2% uranyl acetate and lead citrate and then examined with a Zeiss EM 902 A electron microscope (Zeiss, Oberkochen, Germany).
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