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AMT CCD Camera

Sourced in United States, China

The AMT CCD Camera is a high-performance digital camera designed for advanced microscopy applications. It features a charge-coupled device (CCD) sensor that captures high-resolution images with excellent sensitivity and low noise levels. The camera is capable of capturing detailed images of microscopic samples and is suitable for a wide range of scientific and research applications.

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17 protocols using AMT CCD Camera

Extracellular vesicle (including exosomes and microvesicles) preparation and examination were performed according to previous description (Jung and Mun, 2018 ). LMH cells were cultured to near confluency in a 75 cm dish, and cells were mock infected or infected at a MOI of 1 in serum-free DMEM at 4 °C for 1 h. Then cells were washed once with PBS and cultured in extracellular vesicle-free full DMEM medium in the absence or in the present of PP1 or PP2. Medium was harvested after 24 hpi and extracellular vesicles were purified by differential centrifugation at 4 °C (300×g for 10 min, 2000×g for 20 min, 10000×g for 40 min using a Beckman JA-25.15 fixed angle rotor, then ultracentrifuged 100000×g for 90 min using a Beckman SW-41 swinging bucket rotor). The extracellular vesicle pellets generated were resuspended in serum-free DMEM, fixed and dried. Then samples were coated with gold/palladium alloy by sputter coating and examined under a Hitachi H-7650 transmission electron microscope (Hitachi High Technologies, Shanghai, China), and images were taken using an AMT CCD camera (Advanced Microscopy Techniques, Woburn, MA).
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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).
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3

Immunogold Labeling for Transmission Electron Microscopy

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 minute. For immunogold staining the grids were placed into a blocking
buffer for a block/permeabilization step for 1 hr. Without rinsing, the grids
were immediately placed into the primary antibody at the appropriate dilution
overnight at 4°C (1:300 anti-CD9 ab92726, Abcam and anti-GPC1
PIPA528055, Thermo Scientific). 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 2 hrs at room temperature. Grids were
rinsed with PBS and were placed in 2.5% Glutaraldehyde in 0.1M 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).
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Ventricular specimens (cubes less than 3 mm square) were fixed in 2.5% glutaraldehyde, underwent sectioning and heavy metal uranyl acetate staining for contrast by the Electron Microscopy Center of Indiana University School of Medicine. At least 6 separate sections from each animal strain were analyzed. Electron micrographs were acquired on a transmission electron microscope, Tecnai BioTwin (FEI) equipped with AMT CCD Camera (Advanced Microscopy Techniques).
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Alveolospheres were processed for immunogold labeling described previously (Ridsdale et al., 2011 (link)). Alveolospheres were first fixed in situ with 4% paraformaldehyde (EMS), 0.1% glularaldehyde (EMS), 75 mM L-lysine (Sigma), 10 mM INaO4 (Sigma), and 0.1% CaCl2 in 0.2M HEPES (Sigma), pH 7.2 at room temperature for 10 min, followed by postfixation with fresh fixative at 4 °C overnight. They were embedded with 10% gelatin, cryoprotected with 2.3M Polyvinylpyrrolidone (PVP; M.W. 10,000; Sigma)/sucrose (Sigma) in 0.2M HEPES, pH 7.2, and frozen in liquid nitrogen for cryoultramicrotomy. 70 to 80 nm frozen sections were picked up with mixture of 1.15M PVP/sucrose, 1% methyl cellulose (Sigma), 0.2% uranyl acetate (EMS), and 0.1% glutatraldehyde, transferred to 200 mesh Butvar® coated nickel grids (EMS), and stored at −20 °C until they were ready for immunogold labeling. To localize SFTPB or SFTPC proteins, thawed frozen sections were stained with rabbit polyclonal Ab directed against mature SFTPB (Seven Hills; Lin et al., 1996 (link)) or mature SFTPC (Seven Hills; Ross et al., 1999 (link)), and 10 nm protein A gold (CMC). Electron micrographs of labeled cells were acquired using a Hitachi TEM 7650 (Hitachi High Technologies America) with an AMT CCD camera (Advanced Microscopy Techniques).
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Fixed specimens at an optimal concentration were placed onto 400-mesh carbon/formvar coated grids and allowed to absorb to the formvar for a minimum of 1 min. After rinsing in 1x 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 (TEI) and images were taken with an AMT CCD Camera (Advanced Microscopy Techniques, Woburn, MA, USA).
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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).
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The exosome samples were blotted on a lacey carbon-coated copper grid ‎‎(200-mesh, electron microscopy sciences) using an automated system, Vitrobot ‎‎(FEI, Hillsboro, OR), operated at 100% humidity for 2 seconds. For immunogold labeling, exosomes were first blocked with 0.5% ‎BSA and then successively incubated with mouse anti-glypican antibodies followed by rabbit ‎anti-mouse (Dako, Glostrup, Denmark). The next, samples were incubated by 10-nm ‎gold particles (Cytodiagnostics) for 2 h at room temperature. Samples were then ‎contrasted and embedded in a mixture of 0.4% uranyl acetate and 1.8% ‎methylcellulose. The grids were washed with PBS followed by double distilled ‎water and stained with 0.4% uranyl acetate/1.8% methylcellulose and then dried. ‎Exosomes were observed using a TecnaiTM G2 Spirit BioTWIN transmission electron ‎microscope (TEI) and images were taken with an AMT CCD camera (Advanced ‎Microscopy Techniques, Woburn, MA, USA) at 150 ‎kV. ‎
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Animals were anesthetized and either perfused, or spinal cord segments were fixed in situ for several minutes in cacodylate-buffered tri-aldehyde fixative (57 (link)). Tissue was removed and fixation continued in Kalt's fixative for 24 h. Tissue was post-fixed in buffered 2% osmium tetroxide, dehydrated, and embedded in araldite epoxy resin. Plastic-embedded tissue was sectioned on a Porter-Blum MT-2 ultra-microtome with glass knives for 1-micron sections, or with a diamond knife for thin sections. Thick sections were collected on glass slides and stained with 1% toluidine blue. Thin sections (50–80 nm) were made at selected intervals, collected on bare grids or on Padget-film-supported-1-hole grids, stained with lead-citrate, and examined with a Tecnai G2 12 Bio Twin (FEI, Hillsboro, OR) equipped with AMT CCD Camera (Advanced Microscopy Techniques, Danvers, MA). Two older images were obtained using a Philips 400 transmission electron microscopy.
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After 72-hr macrophage culture, debris and dead cells in the medium were removed by centrifugation at 1,000 × g for 10 min and then filtered through a 0.2-mm filter. The medium was then subjected to ultracentrifugation at 100,000 × g for 4–6 hr at 4°C. After washing with PBS (100,000 × g for 20 min), the Exo-containing pellet was resuspended in PBS.
For electron microscopy, exosomes were fixed with 2% PFA and loaded on Formvar and carbon-coated copper grids. Then the grids were placed on 2% gelatin at 37°C for 20 min, rinsed with 0.15 M glycine and PBS, and the sections were blocked using 1% cold water fish-skin gelatin. Grids were viewed with an FEI Tecnai G2 spirit transmission electron microscope (FEI) and photographed using an AMT CCD camera (Advanced Microscopy Techniques).
Exosome particle size and concentration was determined using NTA with ZetaView PMX 120 (Particle Metrix).
To track exosomes, they were labeled with fluorescent dye using the PKH67 fluorescent cell linker kit (Sigma-Aldrich), according to the manufacturer’s instructions. After labeling, the exosomes were washed in PBS and collected by ultracentrifugation (100,000 × g for 20 min) at 4°C. Then, PKH67-labeled exosomes were resuspended in PBS.
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