For exosome morphology analysis, 5 μl isolated exosome were put on glow discharged carbon coated 400 mesh copper/rhodium grids and stained with 1% uranyl acetate aquous solution. For whole mount immune-electron microscopy, deposit 5μl of 2% paraformaldehyde fixed exosomes on glow discharged formvar-carbon coated copper grids, and let it adsorbed for 20min. After washing with PBS, the grids were incubated with 50mM glycine/PBS for 5min, blocked with 1% coldwater fish skin gelatin (Sigma) for 10min, then incubate with primary antibodies (anti-TSG101, Abcam) in blocking solution for 2 hours at room temperature. Following washing with PBS, gold conjugated secondary antibodies (15nm protein A- gold, Cell Microscopy Center, University Medical Center Utrecht, 35584 CX Utrecht, The Netherlands; 12nm colloidal gold-AffiniPure goat anti-rabbit IgG (H+L), Jackson ImmunoReasearch Laboratories, Inc., West Grove, PA) were applied in the blocking buffer for 1 hour. After washing with PBS, the grids were fixed in 1% glutaraldehyde in PBS for 5min, washed with water, contrasted and embedded in a mixture of 3% uranyl acetate and 2% methylcellulose in a ratio of 1 to 9. All stained grids were examined under Philips CM-12 electron microscope and photographed with a Gatan (4kx2.7k) digital camera (Gatan Inc., Pleasanton, CA)34 (link).
Gatan 4k x2.7k digital camera
Manufactured by Ametek
Sourced in Netherlands
The Gatan (4k x 2.7k) digital camera is a high-resolution imaging device designed for use in electron microscopy applications. It features a 4096 x 2700 pixel sensor, allowing for the capture of detailed and high-quality images. The camera is capable of rapid data acquisition and provides reliable performance for various microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Cm 12 electron microscope, by Thermo Fisher Scientific (6 mentions)
Embed 812, by Electron Microscopy Sciences (5 mentions)
Cm 12 electron microscope, by Ametek (2 mentions)
Fish skin gelatin, by Merck Group (1 mentions)
Anti tsg101, by Abcam (1 mentions)
Spurr resin, by Electron Microscopy Sciences (1 mentions)
Cm12 tem, by Philips (1 mentions)
Embed812 epoxy resin, by Electron Microscopy Sciences (1 mentions)
Cu rh grid, by Ted Pella (1 mentions)
10 protocols using gatan 4k x2.7k digital camera
1
Exosome Morphology and Immune-Electron Microscopy Analysis
2
Transmission Electron Microscopy of Whole Flies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For transmission electron microscopy, whole flies were immersed in 95% ethanol briefly to get rid of any air bubbles, decapitated and immersed into fixative containing 4% glutaraldehyde in 0.1M PIPES buffer, pH 7.2 at room temperature for 2 hours, and then overnight at 4°C. Flies were next embedded in 1% agar and post-fixed with 2% osmium tetroxide with 1.5% potassium ferricyanide in 0.1M PIPES buffer for 1 hour then en block stained with 1% uranyl acetate in ddH2O at 4°C overnight. Samples were dehydrated with ethanol at room temperature before incubation with propylene oxide and embedment in Spurr resin (Electron Microscopy Sciences, Hatfield, PA). 500nm semi-thin sections were stained with 0.1% toluidine blue to evaluate the area of interest. 60nm ultrathin sections were cut, mounted on formvar coated slotted copper grids and stained with uranyl acetate and lead citrate by standard methods. Stained grids were examined under Philips CM-12 electron microscope (FEI; Eindhoven, The Netherlands) and photographed with a Gatan (4k x2.7k) digital camera (Gatan, Inc., Pleasanton, CA).
3
Transmission Electron Microscopy Wound Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transmission electron microscopy (TEM) was carried out in Microscopy Laboratory at NYU Langone Medical Center70 (link). The harvested wounds were dissected (0.5 X1 cm) and put the wounds on top of paper tower. The skin wounds were fixed in the fixative containing 2.5% glutaraldehyde, and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) with 1% tannic acid for 30 min and further dissected to 1 × 3 mm smaller pieces. Fixation process was continued in the same fixative at RT for 2 h, then 4 °C overnight. The skin then post-fixed with 1% osmium tetroxide for 2 h at RT, block staining in 1% uranyl acetate overnight at 4 °C, then dehydration in a standard manner and embedded in EMbed 812 (Electron Microscopy Sciences, Hatfield, PA) for transmission electron microscopy. Semi-thin sections were cut at 1 mm and stained with 1% Toluidine Blue to evaluate the orientation of the sample. Ultrathin sections (60 nm) were cut, mounted on copper grids and stained with uranyl acetate and lead citrate. Stained grids were examined under Philips cm-12 electron microscope (FEI; Eindhoven, The Netherlands) and photographed with a Gatan (4k X2.7 k) digital camera (Gatan, Inc., Pleasanton, CA).
4
Transmission Electron Microscopy of PDGF-Stimulated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Swiss 3T3 cells (4 × 105 per dish) were seeded in a 60-mm dish, and then serum-starved for 16 h. Streptavidin-coated iron oxide nanoparticles (final concentration 2 nM) and biotin-PDGF-BB (final concentration 14 nM) were mixed in serum-free DMEM at 37 °C for 10 min. Cells were stimulated by incubating in the PDGF/nanoparticle-containing DMEM for the indicated times. Cells were then rinsed with PBS and fixed in fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde at room temperature (RT), then scraped and pelleted into microtubes, and consecutively fixed overnight at 4 °C, Cells were post fixed in 1% OsO4, dehydrated in a series of ethanol solutions (30%, 50%, 70%, 85%, 95%, 100%), and embedded in EMbed812 epoxy resin (Electron Microscopy Sciences, Hatfield, PA). 70 nm ultrathin sections were cut, mounted on copper grids and stained with uranyl acetate and lead citrate by standard methods. Grids were viewed using a Philips CM12 TEM (Philips) transmission electron microscope and photographed with Gatan 4k x 2.7k digital camera (Gatan Inc.).
5
Electron Microscopy Sample Preparation
6
Electron Microscopy of Projection Axons
7
Negative Staining Electron Microscopy of Protein Aggregates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Electron microscopy images, using negative staining, to assess the conformational states of monomeric, oligomeric or fibrillar forms of the aggregated and polymerized ABri, p13Bri, Aβ1–40 and Aβ1–42, α-synuclein, PHF and PrP were done as previously described and were taken at the NYULMC OCS Microscopy Core22 (link). Samples were diluted 1 mg/ml in PBS pH 7.4 and vortexed before 3 μl of each one were placed onto carbon coated 400 mesh Cu/Rh grid (Ted Pella Inc., Redding, CA). Negative staining was performed using 1% uranyl acetate diluted in distilled water (Polysciences, Inc, Warrington, PA). Stained grids were examined under Philips CM-12 electron microscope and photographed with a Gatan (4k x2.7k) digital camera. In electron micrographs the pixel size is ~3.3 Å/pixel and the defocus is −0.7µm or −768nm. In most cases samples were kept for weeks or months to repeat the EMs at different times to follow the fibrillization or the stability of the oligomers.
8
Ultrastructural Analysis of Cell Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed in 0.1 M sodium cacodylate buffer (pH 7.2) containing 2.5% glutaraldehyde and 2% paraformaldehyde for 2 hours and post-fixed with 1% osmium tetroxide for 1.5 hours at room temperature, then processed in a standard manner and embedded in EMbed 812 (Electron Microscopy Sciences, Hatfield, PA). Semi-thin sections were cut at 1 μm and stained with 1% Toluidine Blue to evaluate the quality of preservation. Ultrathin sections (60 nm) were cut, mounted on copper grids and stained with uranyl acetate and lead citrate by standard methods. Stained grids were examined under a Philips CM-12 electron microscope (FEI; Eindhoven, The Netherlands) and photographed with a Gatan (4k x2.7k) digital camera (Gatan, Inc., Pleasanton, CA).
9
Ultrastructural Analysis of Ferutinin-Treated Cells
10
Ultrastructural Analysis of Cell Samples
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!