COS-1 cells transiently transfected with KCNC3WT and KCNC3R420H were trypsinized, pelleted, fixed in 1% osmium tetroxide in H2O for 1 h, and then dehydrated with subsequent immersion in 30-100% acetone and embedded in Eponate (Ted Pella). After overnight polymerization at 56°C, samples were stained with methylene blue, counterstained with 5% uranyl acetate in methanol and in Reynold’s lead citrate for 10 min each and viewed with a Jeol 100CX transmission electron microscope at 80kV. Digital images were collected with a XR40 Digital Camera (Advance Microscopy Techniques).
Eponate
Manufactured by Ted Pella
Sourced in United States
Eponate is a specially formulated epoxy resin designed for embedding and cutting thin sections of biological and material science samples. It has a low viscosity and can be easily mixed with hardeners to create a durable and stable embedding medium.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using eponate
1
Transmission Electron Microscopy of WT and Mutant KCNC3
2
Minipig Tissue Preparation for Histological Analysis
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Before fabricating specimens for tissue observation from the experimental animals, a mixture of xylazine HCL (2.3 mg/kg, Bayer) and ketamine (5 mg/kg, Yuhan Corporation) was intravenously injected into the minipigs at four, eight, and 12 weeks after graft to put the minipigs under anesthesia. Then, KCL (2 mmol/1 g, Huons) was swiftly injected into the veins to administer euthanasia and bone fragments including implements and adjacent tissues were collected immediately after the sacrifice.
The tissues collected from two minipigs per each week of collection were used in histomorphometric analysis. The collected tissues were fixed in 70% alcohol for three days and then stained first by leaving them in a Villanueva staining solution for seven to10 days. For dehydration and bleaching, the tissues were left in each of 50, 70, 80, 95, and 100% alcohol solutions for four hours and then left in propylene oxide overnight for complete dehydration. Blocks were made using epoxy resin (Eponate, Ted Pella Inc., Redding, CA, USA) and hardened for three days in an incubator at 60°C. Thereafter, trimmed sections were made using an Accutom-50 (Struers Co., Copenhagen, Denmark) and the trimmed sections were ground using a micro-cutting and grinding system (EXAKT, Exakt Co., Norderstedt, Germany) to make 10-20 μm thick tissue slides.
The tissues collected from two minipigs per each week of collection were used in histomorphometric analysis. The collected tissues were fixed in 70% alcohol for three days and then stained first by leaving them in a Villanueva staining solution for seven to10 days. For dehydration and bleaching, the tissues were left in each of 50, 70, 80, 95, and 100% alcohol solutions for four hours and then left in propylene oxide overnight for complete dehydration. Blocks were made using epoxy resin (Eponate, Ted Pella Inc., Redding, CA, USA) and hardened for three days in an incubator at 60°C. Thereafter, trimmed sections were made using an Accutom-50 (Struers Co., Copenhagen, Denmark) and the trimmed sections were ground using a micro-cutting and grinding system (EXAKT, Exakt Co., Norderstedt, Germany) to make 10-20 μm thick tissue slides.
3
Ultrastructural Analysis of NP Uptake in HeLa Cells
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HeLa cells
were seeded in 6-well plates (2 mL, 5 × 104 cells
per well) and grown for 24 h at standard culture conditions. Then,
200 μL (2.5 × 1010 NP/mL) was added to cells.
After 6 h of incubation, the NP-containing cells were washed three
times with phosphate-buffered saline (PBS), trypsinized, and centrifuged
at 1500 rpm for 4 min. The cell pellets were fixed with 500 μL
of 2.5% (w/v) glutaraldehyde and then included in an agar pellet,
post-fixed with osmium tetraoxide in 0.1 M cacodylate buffer at 1%
(w/v), and, finally, pelletized with Eponate (Ted Pella Inc, Redding,
CA, USA). Ultrathin cuts were obtained with an ultramicrotome (UltraCut
S, Leica Microsystems GmbH) and analyzed with a TEM microscope (JEOL
JEM 1011, Japan).
were seeded in 6-well plates (2 mL, 5 × 104 cells
per well) and grown for 24 h at standard culture conditions. Then,
200 μL (2.5 × 1010 NP/mL) was added to cells.
After 6 h of incubation, the NP-containing cells were washed three
times with phosphate-buffered saline (PBS), trypsinized, and centrifuged
at 1500 rpm for 4 min. The cell pellets were fixed with 500 μL
of 2.5% (w/v) glutaraldehyde and then included in an agar pellet,
post-fixed with osmium tetraoxide in 0.1 M cacodylate buffer at 1%
(w/v), and, finally, pelletized with Eponate (Ted Pella Inc, Redding,
CA, USA). Ultrathin cuts were obtained with an ultramicrotome (UltraCut
S, Leica Microsystems GmbH) and analyzed with a TEM microscope (JEOL
JEM 1011, Japan).
4
TEM Imaging of Neuronal Ultrastructure
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After pretreatment with ginsenoside CK and/or I/R injury, the brains were cut into 2-mm sections and fixed in ice-cold 2.5% glutaraldehyde solution for 15 min. Then, the tissue sections were postfixed in 0.7% potassium ferrocyanide, stained with 2.5% uranyl acetate in 0.1 M maleate, and embedded in Eponate (TedPella, CA, USA). The tissue sections were polymerized overnight and immersed in liquid nitrogen. Thin sections with 60–80 nm were cut with a diamond knife on a Leica EM UC7 ultramicrotome with ultra 45° (Daitome) and collected onto copper grids, which were stained with 4% uranyl acetate in 50% methanol and 5% citrate [23 (link)]. Images were captured with a transmission electron microscope (TECNAI G2 20 TWIN, FEI, Hillsboro, OR, USA).
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