Negative-staining transmission electron microscopy [14 (link)] was performed to establish the morphology of phage Aristophanes. An aliquot of the purified phage preparation was loaded to the carbon-coated copper grid, subjected to glow-discharge, and subsequently negatively stained with 1% uranyl acetate for 30 s and air-dried. Prepared grids were examined using a JEOL JEM-2100 200 kV transmission electron microscope. Images of negatively stained phage particles were taken with a Gatan Ultrascan 1000XP CCD camera and Gatan Digital Micrograph software. The dimensions were averaged among at least 30 individually measured particles.
Ultrascan 1000xp ccd camera
Manufactured by Ametek
Sourced in United States, Japan
The Ultrascan 1000XP is a CCD camera designed for laboratory applications. It features a high-resolution CCD sensor and advanced image processing capabilities. The core function of the Ultrascan 1000XP is to capture and process high-quality digital images for various laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Jem 2100 200 kv, by JEOL (2 mentions)
Digital micrograph software, by Ametek (2 mentions)
Tecnai t12 tem, by Thermo Fisher Scientific (1 mentions)
Jem 1400 electron microscope, by JEOL (1 mentions)
Tacs xl blue label in situ apoptosis detection kit, by Bio-Techne (1 mentions)
Jem 1011 electron microscope, by JEOL (1 mentions)
2100 transmission electron microscope, by JEOL (1 mentions)
Vitrobot filter paper, by Ted Pella (1 mentions)
6 protocols using ultrascan 1000xp ccd camera
1
Morphological Visualization of Phage Aristophanes
2
Imaging Huntingtin Exon1 Fibril Formation
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The fibril morphology and progression of fibril formation by htt exon1 were monitored using negative-stain TEM. Aliquots of sample were diluted with PBS buffer, and then deposited onto freshly glow-discharged carbon-coated copper grids. After removal of excess buffer, grids were treated with negative stain that was adsorbed for 30 s prior to blotting. The 22 and 37 °C exon1 fibrils were stained with 1% (w/v) uranyl acetate and 1% phosphotungstic acid, respectively. The 22 and 37 °C exon1 fibrils in Supplementary Fig. 3 were stained with 1% (w/v) uranyl acetate. Images were obtained at 6,500–30,000-fold magnification using a Tecnai T12 TEM (FEI, Hillsboro, OR) operating at 120 kV and equipped with an UltraScan 1000XP CCD camera (Gatan, Pleasanton, CA). Fibril widths were measured using ImageJ's straight line freehand tool (NIH, Bethesda, MD). Each measurement spanned the length of the negative-stained area of the fibre with similar contrast. Pooled positive stain on the edges of the fibres was not included in the measurements. In images with low resolution, the fibre diameter was determined in regions with the clearest defined boundaries. At least three measurements were obtained per fibre.
3
Ultrastructural Analysis of Mouse RPE
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Mouse eyes were fixed in 4% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) overnight. The tissues were processed as previously described [42 (link)]. One micron plastic sections were stained with toluidine blue. Sixty nanometer thin sections were examined and photographed using a JEM1400 electron microscope (JEOL USA, Peabody, MA) equipped with a Gatan Ultrascan 1000XP CCD camera (Gatan, Pleasanton, CA). Basal laminar deposit (BLamD) severity and frequency in mice were graded based on a semiquantitative grading system described previously [43 (link)].
The thickness of the POS in the toluidine blue–stained sections was measured using the Adobe Photoshop CS4 extended ruler tool at 250 μm interval distances from the optic nerve head. Results of n =3 mice were plotted. POS thickness measurements from the Atg5ΔRPE and Atg7ΔRPE mice were compared with those in the wild-type control mice using the Student t test.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed for the in situ detection of apoptosis on 10 µm cryosections of mouse eyes using a TACS.XL-Blue Label In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD) according to the manufacturer’s instructions.
The thickness of the POS in the toluidine blue–stained sections was measured using the Adobe Photoshop CS4 extended ruler tool at 250 μm interval distances from the optic nerve head. Results of n =3 mice were plotted. POS thickness measurements from the Atg5ΔRPE and Atg7ΔRPE mice were compared with those in the wild-type control mice using the Student t test.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed for the in situ detection of apoptosis on 10 µm cryosections of mouse eyes using a TACS.XL-Blue Label In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD) according to the manufacturer’s instructions.
4
Betulin Sphere Visualization and Vaccine Formulation
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The betulin spheres were diluted to 1 mg/mL in 0.05 M Tris-HCl (pH 7.4) buffer and sonicated at 42 KHz ± 6% 4 times within 30 s. The samples (3 µL) were applied to copper grids and stained with 2% phosphotungstic acid. For electron microscopy, JEOL JEM 1011 electron microscope (“JEOL” Ltd., Tokyo, Japan) was used.
Vaccine samples containing betulin adjuvant (0.4 mg/mL) in DPBS buffer (pH 7.4) with RBD-SD1-Fc protein (40 μg/mL) were sonicated at 42 KHz ± 6% 4 times within 30 s. Then, 3 μL of the samples were applied to carbon-coated glow-discharged copper grids and negatively stained with 1% uranyl acetate two times for 30 s each. Air-dried grids were imaged on a JEOL 2100 transmission electron microscope (“JEOL” Ltd., Tokyo, Japan) equipped with a Gatan Ultrascan 1000XP CCD camera and operated at 200 kV. The data were collected under low-dose conditions at a ~1.5 μm defocus and 40.000 magnification.
Vaccine samples containing betulin adjuvant (0.4 mg/mL) in DPBS buffer (pH 7.4) with RBD-SD1-Fc protein (40 μg/mL) were sonicated at 42 KHz ± 6% 4 times within 30 s. Then, 3 μL of the samples were applied to carbon-coated glow-discharged copper grids and negatively stained with 1% uranyl acetate two times for 30 s each. Air-dried grids were imaged on a JEOL 2100 transmission electron microscope (“JEOL” Ltd., Tokyo, Japan) equipped with a Gatan Ultrascan 1000XP CCD camera and operated at 200 kV. The data were collected under low-dose conditions at a ~1.5 μm defocus and 40.000 magnification.
5
Cryo-EM Preparation of LDL Nanoparticles
6
Negatively Stained Phage Imaging
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The phage was examined by negative contrast electron microscopy, using the following procedure. 3 µL of purified and concentrated phage preparation was applied to the carbon-coated 400 mesh copper grids and subjected to glow-discharge using the Emitech K100× apparatus (Quorum Technologies, Laughton, UK). Grids were then negatively stained with 1% uranyl acetate for 30 s, air-dried, and analyzed using a JEOL JEM-2100 200kV transmission electron microscope. Images of negatively stained phage particles were taken with a Gatan Ultrascan 1000XP CCD camera (14 mkm pixels) and Gatan Digital Micrograph software with the following parameters: 30,000× magnification, 0.5–1 µm defocus, 40 µm objective aperture, 2k × 2k pixel size unbinned image size, 3.4 angstrom pixel size [16 (link)]. At least 30 electronic phage images were used for the phage morphology determination.
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