To analyse the ultrastructure of triple labelled microglia, these cells were sorted on a permeable support PET membrane with 1um size pores (ThinCert™ Cell Culture Inserts, Greiner Bio-One) and prepared for electron microscopy. Fixation was performed in 2.5% (vol/vol) glutaraldehyde (Science Services, Munich, Germany) in 0.2 M sodium cacodylate buffer (Merck, Darmstadt, Germany) at 4 °C overnight. Samples were post-fixed in osmium tetroxide (1%, vol/vol, Serva, Heidelberg, Germany) and stained in 2% uranyl acetate (Science Services, Munich, Germany) in 70% ethanol. After dehydration, membranes were embedded in epoxide resin (Araldite, Serva, Heidelberg, Germany). Finally, blocs were used for ultramicrotomy and sections were stained in 0.4% (vol/vol) lead citrate (Merck, Darmstadt, Germany). Samples were analysed using an EM10 electron microscope (Carl Zeiss, Oberkochen, Germany).
Epoxide resin
Manufactured by Serva Electrophoresis
Sourced in Germany
Epoxide resin is a thermosetting polymer that is commonly used in various industrial applications. It is a clear, viscous liquid that cures to form a hard, durable, and chemically resistant material. The core function of epoxide resin is to serve as a versatile adhesive, sealant, and coating material.
Automatically generated - may contain errors
Lab products found in correlation
Em10 electron microscope, by Zeiss (2 mentions)
Osmium tetroxide, by Polysciences (2 mentions)
Lead citrate, by Serva Electrophoresis (2 mentions)
Glutardialdehyde, by Polysciences (2 mentions)
Lead citrate, by Merck Group (1 mentions)
Thincert cell culture insert, by Greiner (1 mentions)
Uranyl acetate, by Science Services (1 mentions)
Glutaraldehyde, by Science Services (1 mentions)
Osmium tetroxide, by Serva Electrophoresis (1 mentions)
Minispin plus, by Eppendorf (1 mentions)
Matrigel, by Corning (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Reichert ultracut ultramicrotome, by Leica (1 mentions)
Micro star 17, by Avantor (1 mentions)
4 protocols using epoxide resin
1
Ultrastructural Analysis of Microglia
2
Pt-NPPVP Effects on HEI-OC1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For investigation of the effects of Pt-NPPVP on the cellular ultrastructures the HEI-OC1 cells were seeded into a 6-well microtiter plate (Nunclon, Thermo Fisher Scientific, Kempen, Germany) at densities of 300.000 cells per well and cultivated in 3 ml supplemented high glucose DMEM for 24 h as described above. As follows, the cells were cultivated in 3 ml culture medium containing 50–150 μg/ml Pt-NPPVP. Untreated cells in the medium alone served as controls. After 48 hours of incubation, the medium was removed and replaced with PBS. The cells were collected with the help of a cell scraper and centrifuged at 1000 rpm for 4 min (MiniSpin Plus, Eppendorf, Hamburg, Germany). The supernatant was discarded and the cell pellet was fixed with 2.5% glutardialdehyde (Polysciences, Warrington, PA, USA) in 0.1 M sodium cacodylate (Th. Geyer, Hamburg, Germany). After postfixation with 2% osmium tetroxide (Polysciences) in 0.1 M sodium cacodylate the cell pellets were embedded in epoxide resin (Serva, Heidelberg, Germany). Ultra-thin sections stained with 2% uranyl acetate (Serva) and lead citrate (Serva) were examined with the transmission electron microscope (Morgagni 268, 80 kV, Eindhoven, Netherlands). The digital images were processed with Adobe Photoshop CS6.
3
Ultrastructural Analysis of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were seeded on Matrigel (Corning)-coated glass coverslips and cultivated for 2 d before transfection and drug treatment. After washing and fixation with 2.5% glutaraldehyde (Sigma-Aldrich) in 20 mM Hepes buffer (pH 7.4) for 2 h at 37°C, the cells were washed with buffer, post-fixed in 2% osmium tetroxide, dehydrated, and embedded in epoxide resin (Araldite, Serva) as described previously (Wolburg-Buchholz et al, 2009 (link)). Ultrathin sections were performed using a Reichert Ultracut ultramicrotome (Leica) and were analyzed in an EM 10 electron microscope (Zeiss). Images were taken by a digital camera (Tröndle).
4
Platinum Nanoparticle Cellular Ultrastructure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Both cell lines were seeded into a 6-well microtiter plate (Nunclon, Thermo Fisher Scientific, Kempen, Germany) at densities of 3.5 x 105 cells per well and cultivated in 3 ml supplemented high glucose DMEM for 24 h as described above. In order to investigate the effects of Pt–as either nanoparticles or ionic components–on the ultrastructure both cell lines were cultivated in 5 ml culture medium containing either 5–100 μg/ml of Pt-NP or 0.02–6 μg/ml of the Pt-Diss for another 48 h and 6 d, respectively. Additionally, untreated cells–both in the medium alone and in culture medium supplemented with the same NaCl concentration as present in the Pt-Diss solution–were included as controls. The cells were collected using a cell scraper and centrifuged at 1000 rpm for 4 min (Micro Star 17, VWR, Radnor, PA, USA) and fixed with 2.5% glutardialdehyde (Polysciences, Warrington, PA, USA) in 0.1 M sodium cacodylate (Th. Geyer, Hamburg, Germany). After post-fixation with 2% osmium tetroxide (Polysciences) in 0.1 M sodium cacodylate the cell pellets were embedded in epoxide resin (Serva, Heidelberg, Germany). Ultra-thin sections stained with 2% uranyl acetate (Serva) and lead citrate (Serva) were examined under the transmission electron microscope (Tecnai G2 200 kV TEM, Eindhoven, Netherlands). The digital images were processed using Adobe Photoshop CS6.
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!