The eyes were enucleated, and a slit was cut into the cornea to allow for rapid influx of fixative. Eyes were fixed overnight in 2.5% glutaraldehyde, 1% formaldehyde, 3% sucrose, and 1 mM MgSO4 in 0.1 molar (M) phosphate buffer, pH 7.4. The eyes were then dissected and small central, nasal portions were osmicated for 60 minutes in 0.5% OsO4 in 0.1 M phosphate buffer, processed in maleate buffer for en bloc staining with uranyl acetate, dehydrated in graded ethanols, and processed for resin embedding as published.30 (link) Serial sections were cut at 90 nm on a Leica Ultramicrotome, Leica Biosystems Division of Leica Microsystems Inc. Buffalo Grove, IL, USA, onto carbon-coated Formvar films, Ted Pella, Inc. Redding, CA, USA, supported by nickel slot-grids.
Formvar film
Manufactured by Ted Pella
Sourced in United States
Formvar film is a thin, transparent material used as a support film for electron microscopy samples. It serves as a substrate to hold and protect the specimen during the imaging process. Formvar film is made from a synthetic polymer and is designed to be durable, stable, and have low electron scattering properties to minimize interference with the electron beam.
Automatically generated - may contain errors
Lab products found in correlation
Ultramicrotome, by Leica (1 mentions)
Tecnai g2 tem microscope, by Thermo Fisher Scientific (1 mentions)
1200 ex 2 electron microscope, by JEOL (1 mentions)
Tecnai g2 spirit biotwin electron microscope, by Thermo Fisher Scientific (1 mentions)
Pelco easiglow, by Ted Pella (1 mentions)
Veleta ccd camera, by Olympus (1 mentions)
1200ex 2 electron, by JEOL (1 mentions)
Eponate 12, by Ted Pella (1 mentions)
Uc7 ultramicrotome, by Leica (1 mentions)
7 protocols using formvar film
1
Ultrastructural Analysis of Retinal Tissue
2
Visualizing polyplexes by TEM
3
Palladium Nanoparticle Characterization
4
Negative Staining of Biomolecules for TEM
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400 mesh copper grids coated with a carbon film, overlayed with a Formvar® film (TedPella, Inc., CA, USA) were hydrophilized using a glow discharger (Pelco EasiGlow™, TedPella Inc., CA, USA). A glow discharged grid was mounted on tweezers and 3 μL of sample were placed onto the grid. After an adsorption time of ca. 10 sec, the excess of liquid present on grid was blotted-off using Grade 1 filter paper pre-wetted with 12 μL of water and immediately washed with 3 μL of distilled water. Excessive liquid was then blotted-off after ca. 10 sec and 3 μL of Uranyl acetate 2% (Electron Microscopy Sciences, PA, USA) was immediately added to the grid and blotted-off after ca. 10 sec. The grid was then inserted in a Tecnai G2 Spirit BioTwin electron microscope (FEI, OR, USA) equipped with a tungsten filament operating at 100 kV. The grid was visually screened and areas suitable for imaging were identified. Representative images were subsequently acquired at a resolution of 2048 x 2048 pixels using a Veleta CCD camera (Olympus, Tokyo, Japan).
5
TEM Imaging and Particle Size Analysis
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Samples of ZIF-8 nanoparticles diluted in ethanol were loaded onto 200 mesh carbon-coated copper grids with Formvar film (Ted Pella, Inc.) and allowed to air dry before imaging. TEM images were obtained using a JEOL 1200 EX II electron microscope operating at 100 kV. Particle size distribution analysis was conducted using ImageJ software.
6
Characterization of Cellulose Nanocrystals by TEM
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A CNC sample (freeze-dried for long term storage from an aliquot CNC colloid generated in the previous study [31 (link)]) was re-suspended in water (0.1 g in 100 mL H2O, 0.1% w/v) and sonicated for 15 min. A droplet of the CNC colloid was mounted on a copper grid (300 mesh) coated with formvar film (Ted Pella Inc., Redding, CA, USA) for 20–30 min. Excess liquid was removed with filter paper and the grid was negatively stained with phospho-tungstic acid (2% w/v) for 15 s. Excess stain was removed with filter paper and the copper grid was loaded on a TEM operating at 80 kV electron speed and 110,000× magnification. Length and width of well separated CNC rods (total count of 75 from all triplicates), with distinguishable tip ends and widths, were analysed using image J software (National Institute of Health, Rockville, MD, USA). A straight line tool, standardized by the scale bar generated from the TEM micrograph, was used for manual measurement of individual particles on the software. The aspect ratio of an individual CNC particle was calculated from the ratio of the longest side to the shortest side.
7
Ultrastructural Analysis of Mouse Brain
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Two three months old C57Bl6/J mice (one male and one female) were perfusion fixed through the heart with MORF fixative (2.5% glutaraldehyde, 1% paraformaldehyde in 0.1M PIPES buffer, pH 7.4), the brain was dissected out and postfixed in MORF at +4 C overnight. Coronal vibratome sections (200 mm) of the cerebral cortex were generated and stored in MORF until embedding in LR white. Vibratome sections were processed 10 min in 0.1M phosphate buffer, pH 7.4; 60 min in 1% osmium tetroxide in 0.1% phosphate buffer, pH 7.4; 10 min in 50% EtOH; 10 min in 70% EtOH; 10 min in 95% ETOH; 15 min in 100% EtOH; 5 min in propylene oxide; 60 min in propylene oxide:Eponate 12Ô (Ted Pella); overnight in Eponate 12Ô; cut in smaller pieces and transferred to tightly sealed gelatin capsules filled with Eponate 12Ô and incubated at 60 C for 48h to polymerize. 1 mm sections were generated and stained with Toluidine blue to identify region of interest. Finally, ultra-thin sections (60 nm) were cut on a UC7 ultramicrotome (Leica) and collected on nickel mesh grids coated with a Formvar film (Ted Pella). Sections were analyzed in a TecnaiÔ G2 Spirit BioTwin at 80 kV.
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