WT and TAZ-KO mouse neutrophils were cultured with and without estrogen for four days. Cultured neutrophils were gently pelleted, then fixed in 0.15 M cacodylate buffered 3% glutaraldehyde with 2 mM CaCl2 overnight at ambient temperature. Samples were rinsed in buffer then immersed in cacodylate buffered 2% OsO4 for 90 minutes. Samples were rinsed in water, then stained enbloc with 1% Uranyl acetate overnight at 4 C, rinsed in water, dehydrated in a graded series of ethanol to 100%, then transferred to 100% acetone. Samples were infiltrated with Spurrs’ epoxy and polymerized overnight at 70 C. Sections 80 nm in thickness were collected onto formvar coated slot grids, stained in uranyl acetate followed by lead citrate and examined using a FEI G20 TEM operated at 120KV.
G20 tem
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The G20 TEM is a transmission electron microscope (TEM) designed and manufactured by Thermo Fisher Scientific. It provides high-resolution imaging and analysis capabilities for a wide range of materials and applications. The core function of the G20 TEM is to enable researchers and scientists to visualize and study the internal structure and composition of samples at the nanoscale level.
Automatically generated - may contain errors
Lab products found in correlation
Tannic acid, by Merck Group (4 mentions)
Paraformaldehyde (pfa), by Tousimis (3 mentions)
Epoxy tissue stain, by Electron Microscopy Sciences (1 mentions)
Jem 2100 tem, by JEOL (1 mentions)
F 2700 fluorescence spectrophotometer, by Hitachi (1 mentions)
Uv 160a spectrophotometer, by Shimadzu (1 mentions)
X pert pro diffractometer, by Malvern Panalytical (1 mentions)
Spectrum one spectrometer, by PerkinElmer (1 mentions)
7 protocols using g20 tem
1
Estrogen Effects on Neutrophil Ultrastructure
2
Ultrastructural Analysis of Mouse Hindlimb Collagen
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Mouse hindlimbs were fixed in freshly prepared 1.5% glutaraldehyde/1.5% paraformaldehyde (Tousimis) with 0.05% tannic acid (Sigma) in serum-free Dulbecco’s Modification of Eagle’s Medium (DMEM) at 4 °C overnight. Following dissection, samples were post-fixed in 1% OsO4, rinsed in DMEM and dehydrated in a graded series of ethanol to 100%. Samples were then rinsed in propylene oxide, infiltrated in Spurrs epoxy, and polymerized at 70 °C overnight. TEM images were acquired using a FEI G20 TEM at multiple magnifications to visualize transverse sections of collagen fibrils. Collagen fibril diameter was measured using Bioquant Osteo II (Nashville, TN).
3
Ultrastructural Analysis of Mouse Tendons
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Mouse hindlimbs were fixed in freshly prepared 1.5% glutaraldehyde/1.5% paraformaldehyde (Tousimis) with 0.05% tannic acid (Sigma) in DPBS at 4 °C with agitation overnight. The following day, dissect the relevant tendons out of the limb in PBS as follows: for the Achilles, take the mid-tendons that lies below the myotendinous junction and above the enthesis (~ 1–1.5 mm in length); and for the patellar tendon, take the complete mid-tendon between the patella and tibial enthesis. Following dissection, samples were post-fixed in 1% OsO4, rinsed in DMEM and dehydrated in a graded series of ethanol to 100%. Samples were then rinsed in propylene oxide, infiltrated in Spurrs epoxy, and polymerized at 70 °C overnight. TEM images were acquired using a FEI G20 TEM at multiple magnifications to visualize transverse sections of collagen fibrils. Collagen fibril diameter was measured using ImageJ. TEM images of tendons from 3 mice per group were analyzed. Total 545 control fibrils and 905 Rcn3 mutant fibrils from patellar tendons were counted. Total 1568 control fibrils and 1871 Rcn3 mutant fibrils from Achilles tendons were counted.
4
Ultrastructural Analysis of TMJ Condyles
5
Quantitative Analysis of Collagen Fibrils
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The collagen fibril analysis was previously described43 (link). Briefly, 3D tendon constructs were harvested and fixed in the fresh buffer (1.5% glutaraldehyde/1.5% paraformaldehyde (Tousimis) with 0.05% tannic acid (Sigma) in DPBS) at 4 °C overnight. The isolated 3D tendon constructs were post-fixed in 1% OsO4 and rinsed in DMEM. The samples were then gradually dehydrated in a series of ethanol to 100%. Finally, samples were rinsed in propylene oxide, infiltrated in Spurrs epoxy, and polymerized at 70 °C overnight. A FEI G20 TEM was used for transmission electron microscopy (TEM) images which visualize transverse sections of collagen fibrils at multiple magnifications. Collagen fibril diameter was measured using ImageJ software (Fiji 2.15.0). TEM images of six constructs per group were analyzed. Total of 3452 fibrils in vehicle-treated constructs and 3507 fibrils in TGFβ1-treated constructs were counted.
6
Comprehensive Characterization of Nanoparticles
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Fourier transform infrared spectrophotometric (FTIR) spectra were measured on a Spectrum One spectrometer (PerkinElmer, USA) using the KBr pellet technique. Power X-ray diffraction (XRD) measurements were performed on a X'Pert PRO diffractometer (PANalytical, Holland). Thermogravimetric analysis (TGA) was performed on a TGA/SDTA85le instrument (METTLER TOLEDO, Switzerland) with a heating rate of 10 °C min−1 under a nitrogen flow. Zeta potentials were determined by a Nano-zs90 laser particle size and zeta potential analyzer (Malvern, UK). UV-vis spectra were measured on a UV-160A spectrophotometer (Shimadzu, Japan). The morphology and the mesoporous structure of the nanoparticles were confirmed on JEM-2100 TEM (JEOL, Japan) and G20 TEM (FEI, USA). The fluorescence spectra were carried out with a F2700 fluorescence spectrophotometer (Hitachi, Japan). The Laser used an 808 nm NIR laser equipment (Changchun New Industries electronics Tech Co., Ltd, China). The temperature of suspension was monitored by a thermocouple.
7
Ultrastructural Analysis of Mouse Joint Tendons
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Mouse ankle and knee joints were dissected and fixed in fresh 1.5% glutaraldehyde/1.5% PFA (Tousimis) with 0.05% tannic acid (Sigma) in 1 × PBS at 4°C overnight to preserve the native tension on relevant tendons. The next day, flexor digitorum longus (FDL), Achilles, and patellar tendons were dissected out in 1 × PBS, and placed back into fixative. Samples were then post-fixed in 1% osmium tetroxide (OsO4), rinsed in Dulbecco’s Modified Eagle Medium (DMEM), and dehydrated in a graded series of ethanol to 100%. Samples were rinsed in propylene oxide, infiltrated in Spurrs epoxy, and polymerized at 70°C overnight. TEM images were acquired using an FEI G20 TEM at multiple magnifications to visualize transverse and longitudinal sections of collagen fibrils. Collagen fibril diameter was measured using the Fiji release of ImageJ (Schindelin et al., 2012 (link)).
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