To verify the nature of the HtrA1-generated fibrils as amyloid, thioflavin T fluorescence was recorded as a function of time. FAS1-4 protein was incubated with and without the presence of HtrA1 using a final ThT concentration of 40 μm (Ex 450 nm, Em 480 nm). The samples were then further analyzed by FTIR spectroscopy on a Tensor 27 (Bruker) FTIR spectrophotometer equipped with a DTGS Mid-IR detector and a Golden Gate single-reflection diamond-attenuated total reflectance cell (Specac). Samples were placed on the ATR crystal and dried using dry nitrogen. Spectra were recorded from 4000–1000 cm−1 using a resolution of 2 cm−1 and 64 accumulations. The data were corrected for background and atmospheric interference and normalized to the peak intensity. Finally, TEM micrographs were taken for all samples. Five μl of sample were mounted on 400 mesh carbon-coated, glow-discharged nickel grids for 1 min, stained with 1 drop of phosphotungstic acid (PTA), pH = 7.0 and blotted dry on filter paper. EM was performed on a JEOL-1010 electron microscope (JEOL, Tokyo, Japan) operated at 60 keV. The microscope was coupled to an electron-sensitive CDC camera (KeenView, Olympus Soft Imaging Solutions GmbH, Münster, Germany). A grid-size replica (2160 lines/mm) was used for size determination.
Keenview
Manufactured by Olympus
Sourced in United States, Germany, Japan
KeenView is a high-performance digital microscope camera from Olympus. It captures detailed images and videos with a range of resolutions and frame rates to suit various applications. The camera features a CMOS sensor and advanced image processing capabilities to provide clear, high-quality results.
Automatically generated - may contain errors
Lab products found in correlation
Tecnai g2 spirit, by Thermo Fisher Scientific (2 mentions)
Cm120, by Thermo Fisher Scientific (2 mentions)
Quemesa, by Olympus (2 mentions)
Jem 1010, by JEOL (2 mentions)
1010 electron microscope, by JEOL (1 mentions)
Single reflection diamond attenuated total reflectance cell, by Specac (1 mentions)
Tensor 27, by Bruker (1 mentions)
Ftir spectrophotometer, by Bruker (1 mentions)
Jem 2100, by JEOL (1 mentions)
Fei cm120 electron microscope, by Thermo Fisher Scientific (1 mentions)
Anti cd9 antibody, by BD (1 mentions)
Phosphotungstic acid, by Ted Pella (1 mentions)
Jem 1010, by Olympus (1 mentions)
11 protocols using keenview
1
Amyloid Characterization of HtrA1 Fibrils
2
Transmission Electron Microscopy for Actin Filament Imaging
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Specimens were observed under a transmission electron microscope (JEM-2100, JEOL, Tokyo) operated at 80 kV. Images were recorded with a charge-coupled device (CCD) camera (KeenView, Olympus Soft Imaging Solutions, Tokyo) under the control of a personal computer. Typically, the gold pattern was first located at a low magnification to locate the target region, then returned to a normal magnification mode to observe the actin filaments. Image processing, such as trimming, rotation, and enhancement of contrast, were performed using ImageJ, an image processing program operated using a Windows platform [20 (link)].
3
Immunogold Labeling of ABCB6 and CD63
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In EM studies, ABCB6 was revealed by the monoclonal antibody (Santa Cruz Biotechnology); the antibody recognizing CD63 was kindly provided by E. Rubinstein (Université Paris-Sud, France) [29 (link)]. For ultrathin cryosectioning and immunogold-labelling, cells were fixed with a mixture of 2% PFA and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Cells were processed for ultracryomicrotomy and single or double immunogold-labelled using PAG10 (Protein A–gold, 10 nm) or PAG15 as described [30 (link)]. All samples were analysed using a FEI CM120 electron microscope (FEI Company) and digital acquisitions were made with a numeric camera (Keen View; Soft Imaging System). The definition of the distinct compartments was based on their morphology and by correlation with immunogold-labelling for CD63 as a marker of late endosomes/lysosomes. Multivesicular bodies were defined as compartments delimited by a membrane with numerous internal vesicles. Electron-dense compartments with vesicular or lamellar membranes were classified as mixed lysosomes. Electron-dense compartments with no internal membranes were classified as dense lysosomes.
4
Quantifying Melanocyte Melanosome Stages
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Control- and peptide-treated MNT-1 cells grown on coverslips for 3 days—in media containing or not 10 µM peptides (media were renewed every day)—were fixed with 2.5% glutaraldehyde/0.1 M cacodylate buffer, pH 7.2, post-fixed with 1% OsO4 supplemented with 1.5% potassium ferrocyanure, dehydrated in ethanol, and embedded in Epon. Ultrathin sectioning was performed with an ultramicrotome (UCT, Leica, Wetzlar, Germany). Sections were counter-stained with uranyl acetate and lead citrate and electron micrographs were acquired under electron microscopes (Philips CM120 or Tecnai Spirit G2; FEI, Eindhoven, The Netherlands) equipped with a numeric camera (Keen View; Soft Imaging System) or 4k CCD camera (Quemesa, Olympus, Muenster, Germany). Melanosome stages from I to IV were quantified based on their morphology and melanin content (see Table 2 and [3 (link)]). Two independent quantifications by two different authors were performed on more than 100 structures. Results presented in Figure 2 c: Stage I: H2O, 4 ± 3%; QK-5, 5 ± 3%; Stage II: H2O, 11 ± 7%; QK-5, 17 ± 11%; Stage III: H2O, 31 ± 8%; QK-5, 52 ± 8%; Stage IV: H2O, 55 ± 11%; QK-5, 26 ± 16%.
5
Ultrastructural Analysis of Melanocytes
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For conventional EM, MNT-1 cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (1.5 h on ice), postfixed with 1% OsO4/1.5% potassium ferricyanide (45 min on ice), dehydrated in ethanol, and embedded in epon resin. For ultrathin cryosectioning, MNT-1 cells fixed with 2% PFA and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) were processed for ultracryomicrotomy and immunogold labeled using TYRP1 antibody (TA99) followed by protein A conjugated to 10 nm gold (PAG10). Electron micrographs were acquired using Philips CM120 or Tecnai Spirit G2 (FEI) equipped with a numeric camera (Keen View; Soft Imaging System) or 4k charge coupled device camera (Quemesa; Olympus).
6
Transmission Electron Microscopy of MSN
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Hitachi transmission electron microscope (Hitachi High-technologies, Tokyo, Japan), model H-8100, with a LaB6 filament (Hitachi High-Technologies Europe GmbH, Krefeld, Germany) complemented with an accelerator voltage of 200 kV and a current of 20 µA. A camera KeenView (Soft Imaging System, Münster, Germany) is incorporated in this equipment, which through the iTEM software allows acquiring TEM images. MSN dispersed in ethanol were prepared and dried on a Formvar carbon-coated copper grid 200 mesh (Ted Pella, Redding, CA, USA).
7
Transmission Electron Microscopy of RPT Aggregates
8
Immunoelectron Microscopy of Extracellular Vesicles
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Fractions 8 and 9 were pooled and up-concentrated using a 10 kD spin filter at 14,000×g at 4°C. Immunolabelling was performed by mounting concentrated samples on carbon-coated, glow discharged 400 mesh Ni grids for 30 s and washed 3 times with PBS. Grids were blocked with 0.5% ovalbumin in PBS and then incubated with primary anti-CD9+ antibody (BD Biosciences, Albertslund, Denmark, #555370) 1:50 in 0.5% ovalbumin in PBS for 30 min at 37°C. After incubation grids were washed 3 times with PBS and incubated with secondary antibody goat antimouse conjugated with 10 nm colloidal gold (British BioCell, Cardiff, UK) 1:20 in 1% cold fish gelatine. Samples and secondary antibody were incubated for 1 h at 37°C. The grids were then washed with 3 drops of PBS, before applying 2 drops of 1% cold fish gelatin for 10 min each. The grids were finally washed with 3 drops of PBS before staining with 2 drops of 1% (W/V) phosphotungstic acid at pH 7.0 and blotted dry. Images were obtained with a transmission electron microscope (JEM-1010, JEOL, Eching, Germany) operated at 60 keV coupled to an electron-sensitive CCD camera (KeenView, Olympus, Center Valley, PA, USA). For size determination of visible EVs a grid-size replica (2,160 lines/mm) was used.
9
Characterization of Extracellular Vesicles by TEM
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EVs were phenotypically and structurally characterized by Transmission Electron Microscopy (TEM) with immuno-gold labelling against CD9, as previously described by Nielsen et al [44 (link)]. Briefly, 5 μl of EV isolate was mounted on a grid (SPI Supplies, PA, USA) and stained with one drop of 1% (w/v) phosphotungstic acid (pH 7.0, Ted Pella, Caspilor AB, Sweden), and subsequently blotted dry on filter paper. To visualize the presence of EV-specific marker CD9 on the surface of vesicles, IEM was performed on the isolated vesicles. The pelleted vesicles were positioned on a grid as described above and then blocked in ovalbumin. Subsequently, the grid was incubated with primary anti-CD9 antibody (1:50, BD Pharmingen, CA, USA), followed by incubation with secondary goat anti-mouse antibody conjugated with 10 nm colloidal gold (1:25, British BioCell, UK). The grids were stained with 1% (w/v) phosphotungstic acid at pH 7.0 and blotted dry. Images were obtained with a transmission electron microscopy (JEM 1010, Germany) operated at 60 keV coupled to an electron-sensitive CCD camera (KeenView, Olympus, PA, USA). Lastly, a grid-size replica (2,160 lines/mm) was imported to ImageJ 1.50i software, which enables a correct determination of the size of EVs.
10
Visualizing Exosome Morphology via TEM
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The shape of EVs was examined using transmission electron microscopy (TEM) employing a E1610 system (Weihui Biotechnology, Beijing, China). Initially, a 10-μL drop of exosome suspension was placed carefully onto a parafilm membrane. Subsequently, the exosome-coated membrane was covered with a formvar carbon membrane and left undisturbed for 10 min to facilitate the maximum adsorption of exosomes. After application of a wash buffer, TEM was used to visualize exosomes ( 25 ). Images were captured using an electron-sensitive charge-coupled device camera (KeenView; Olympus).
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