Grids for negative staining were generated by applying 3 μl of sample (~0.1–1 mg/ml) on to carbon-coated 400 mesh copper grids (EM Resolutions). Prior to applying the sample, grids were glow-discharged for 20 s at 10 mA (Leica EM ACE200). Excess liquid was removed, and the grid was stained with 2% (w/v) uranyl acetate for 30 s. Grids were viewed using a FEI Tecnai G2 20 TWIN or FEI Talos 200 C TEM (FEI UK Ltd) at 200 kV and imaged using either an AMT XR-60 or OneView 4k × 4k digital camera (Gatan).
Oneview 4k 4k digital camera
Manufactured by Ametek
Sourced in United Kingdom
The OneView 4k × 4k digital camera is a high-resolution imaging device capable of capturing detailed images at a resolution of 4096 × 4096 pixels. It is designed for applications that require precise and accurate visual data.
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3 protocols using oneview 4k 4k digital camera
1
Negative Staining for Transmission Electron Microscopy
2
TEM Tissue Ultrastructural Analysis
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Samples were fixed in Karnovsky’s fixative: 2% Glutaraldehyde (EMS Cat# 16000) and 4% paraformaldehyde (EMS Cat# 15700) in 0.1M Sodium Cacodylate (EMS Cat# 12300) pH 7.4 for 1 hr. The fix was replaced with cold/aqueous 1% Osmium tetroxide (EMS Cat# 19100) and were then allowed to warm to Room Temperature (RT) for 2 hrs rotating in a hood, washed 3X with ultrafiltered water, then en bloc stained in 1% Uranyl Acetate at RT 2hrs while rotating. Samples were then dehydrated in a series of ethanol washes for 30 minutes each @ RT beginning at 50%, 70% EtOH then moved to 4°C overnight. They were place in cold 95% EtOH and allowed to warm to RT, changed to 100% 2X, then Propylene Oxide (PO) for 15 min. Samples are infiltrated with EMbed-812 resin (EMS Cat#14120) mixed 1:2, 1:1, and 2:1 with PO for 2 hrs each with leaving samples in 2:1 resin to PO overnight rotating at RT in the hood. The samples are then placed into EMbed-812 for 2 to 4 hours then placed into molds w/labels and fresh resin, orientated and placed into 65° C oven overnight.
Sections were taken around 80nm using an UC7 (Leica, Wetzlar, Germany) picked up on formvar/Carbon coated 100 mesh Cu grids, stained for 40s in 3.5% Uranyl Acetate in 50% Acetone followed by staining in Sato’s Lead Citrate for 2 minutes. Observed in the JEOL JEM-1400 120kV. Images were taken using a Gatan OneView 4k × 4k digital camera.
Sections were taken around 80nm using an UC7 (Leica, Wetzlar, Germany) picked up on formvar/Carbon coated 100 mesh Cu grids, stained for 40s in 3.5% Uranyl Acetate in 50% Acetone followed by staining in Sato’s Lead Citrate for 2 minutes. Observed in the JEOL JEM-1400 120kV. Images were taken using a Gatan OneView 4k × 4k digital camera.
3
TEM Tissue Ultrastructural Analysis
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