Cmias 2001B multifunctional color pathological image analysis system (Beihang University, China); IM1000 ECG monitor (COULD Inc, the United States); JE M1010 transmission electron microscopy (TEM) (JEOL Inc, Japan); Twin Block PCR thermal cycle system (ERICOMP Inc, the United States); GAA7001B gel image analysis system (UVI Inc, the United Kingdom). Fraxiparine (Low-Molecular-Weight Heparin Sodium Injection, 9500 IU anti-Ⅹa/ml according to European pharmacopoeia, Sanofi Pharma, France). Rabbit anti-rat PDG -B polyclonal antibody (Wuhan Boster Biotechnology Company, China).
Je m1010 transmission electron microscopy
Manufactured by JEOL
Sourced in Japan
The JEM-1010 is a transmission electron microscope (TEM) manufactured by JEOL. It is designed to provide high-quality imaging of microscopic samples. The JEM-1010 TEM utilizes an electron beam to illuminate and magnify samples, allowing users to observe the internal structure and morphology of materials at the nanometer scale.
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4 protocols using je m1010 transmission electron microscopy
1
Multifunctional Pathological Image Analysis
2
Ultrastructural Visualization of Autophagy
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HMECTERT/ST/ER-RasV12 cells were seeded onto 4-chambered coverglass (Lab-Tek Chambered Coverglass System) (Nalgene-Nunc, Rochester, NY, USA) and incubated in medium without EGF, insulin, and hydrocortisone for 24 hr to stimulate autophagy. Samples were fixed in 2.5% glutaraldehyde in PBS at 4°C for 1 hr before osmication with 1% osmium tetroxide, pH7.4 for 1 hr. Subsequently the samples were dehydrated through an ascending series of ethanol at room temperature before infiltration with acetone and resin, followed by final embedding in resin which was allowed to polymerise at 60°C for 24 hr. Samples were cut by an ultra-microtome (Leica), mounted on formvar-coated copper grids and stained with lead citrate. The grids were viewed in a JEOL JEM 1010 transmission electron microscopy (TEM).
3
Characterizing Lipoprotein Nanoparticle Formulations
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The average particle diameter, particle size distribution, and surface charge were determined by dynamic light scattering (DLS) technique using a Zetasizer Nano ZS (Malvern, UK).23 (link) The morphology and size of the particles were observed using JEM-1010 transmission electron microscopy (JEOL, Tokyo, Japan). The encapsulation capacity of LPNs was determined by measuring the concentrations of CBP and PTX using a C-18 reverse-phase high-performance liquid chromatography (RP-HPLC) (Beckman Coulter, Brea, CA) at a flow rate of 1 mL/min.24 (link) Tert-butyl methyl ether (5 mL) was added to the LPNs suspension and vortex for 1 min to extract the drugs and then redissolved in acetonitrile:water (10:90) solution. The solution (1 mL) was injected into the C-18 column, and CPT and PTX were detected at 227 nm after different retention times. The amounts of CBP and PTX were quantified by HPLC. The encapsulation efficiency (EE) was determined as: (Amount of entrapped drug/amount of total drug) × 100%.
4
Pancreatic Tissue Analysis: Histology and Ultrastructure
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Small pieces were obtained from pancreatic tail, fixed in 10% buffered formalin and processed for examination by light microscopy that were subjected to [32 33 ].
1- Hematoxylin and eosin (H&E).
2- Immunohistochemical staining for detection of insulin antibody using the avidin–biotin peroxidase system.
The primary antibodies used were mouse monoclonal insulin antibodies (Medico Company, Egypt) at a dilution of 1:100 that incubated with slides for 1 h at room temperature. Then, the sections were counterstained with Meyer's hematoxylin.
For electron microscopy, thin slices of pancreas were fixed in 5% cold glutaraldehyde then washed three times with phosphate buffer (pH 7.2) and post fixed in cold osmium tetroxide for 2 h. Subsequently, the specimens were washed in buffer, dehydrated in a graded series of alcohol and embedded in Epon. Ultrathin sections (50–80 nm) were cut with ultramicrotome, collected on copper grids and stained with uranyl acetate and lead citrate to be examined [34 ] by (JEM-1010) transmission electron microscopy (TEM; Jeol, Tokyo, Japan) in Tanta University Electron Microscopy Unit.
1- Hematoxylin and eosin (H&E).
2- Immunohistochemical staining for detection of insulin antibody using the avidin–biotin peroxidase system.
The primary antibodies used were mouse monoclonal insulin antibodies (Medico Company, Egypt) at a dilution of 1:100 that incubated with slides for 1 h at room temperature. Then, the sections were counterstained with Meyer's hematoxylin.
For electron microscopy, thin slices of pancreas were fixed in 5% cold glutaraldehyde then washed three times with phosphate buffer (pH 7.2) and post fixed in cold osmium tetroxide for 2 h. Subsequently, the specimens were washed in buffer, dehydrated in a graded series of alcohol and embedded in Epon. Ultrathin sections (50–80 nm) were cut with ultramicrotome, collected on copper grids and stained with uranyl acetate and lead citrate to be examined [34 ] by (JEM-1010) transmission electron microscopy (TEM; Jeol, Tokyo, Japan) in Tanta University Electron Microscopy Unit.
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