Electron microscopy specimens were prepared by submerging a Formvar carbon-coated copper grid (Electron Microscopy Sciences) in 10 μl of protein solution and incubating it for 5 min. These grids were then stained with 10 μl of a 1% uranyl acetate solution for 2 min at room temperature. The grids were then tapped on two additional 10 μ1 uranyl acetate drops and finally, rinsed in deionized water. Filter paper was used to remove the excess of stain and water before imaging. The negatively stained samples were examined for droplets or fibrils with a JEOL JEM-1400 TEM electron microscope. Images were acquired using a Gatan Orius digital camera at magnification of 5000–10,000×. Fibril images were analyzed using ImageJ including the FibrilJ plugin (45 ).
Jem 1400 tem electron microscope
Manufactured by JEOL
Sourced in Japan
The JEM-1400 is a transmission electron microscope (TEM) manufactured by JEOL. It is designed to provide high-resolution imaging and analysis of samples at the nanoscale level. The JEM-1400 utilizes an electron beam to illuminate and interact with the specimen, allowing for the observation of fine structural details and the examination of materials at the atomic scale.
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4 protocols using jem 1400 tem electron microscope
1
Negative Staining for TEM Imaging
2
Virus Imaging using Electron Microscopy
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Formavar-coated nickel grids were floated on drops (20 µl) of purified virus preparation for 5 min. After rinsing with distilled water, the grids were stained with 2% uranyl acetate and examined with a Jeol JEM-1400 TEM electron microscope, Faculty of Science—Alexandria University.
3
Ultrastructural Characterization of Telocytes
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Immediately following surgical excision of the rat bladder, the bladder specimens were cut into 1 × 1 mm pieces, fixed in 2.5% glutaraldehyde in cacodylate buffer for 2 h at 4oC. The tissues were then washed in cacodylate-sucrose buffer and post fixed for 1 h at 4oC in 2% osmium tetroxide. After dehydration in graded ethanol, the samples were impregnated in Epon 812 substitute (EMBed-812 Kit, Electron Microscopy Science, USA) at room temperature, and polymerised at 60 °C for 48 h. Semi-thin sections were cut, stained with methylene blue-azure II, and examined by light microscopy to choose the region of interest for ultrathin sectioning. The ultrathin sections were then prepared using an Ultracut R ultramicrotome (Leica, Vienna, Austria), double stained with uranyl acetate and lead citrate. Cellular ultrastructural morphological characterisation was examined at 80 kV with a Jeol TEM Jem-1400 electron microscope (Jeol, Japan). Identification and differentiation of telocytes and Cajal was done according to the description in previous works. We examined each case and counted telocytes and/or telopodes (TPs) in 3 examined TEM fields.
4
Paraffin-embedded TEM Tissue Sampling
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Transmission electron microscopy (TEM) studies were performed using a retrospective technique similar to that used for tissue microarray to select specific areas of formalin-fixed, paraffin-embedded blocks25 (link). After matching the histological and IHC tissue sections to the paraffin block, extraction of cylindrical tissue cores containing the area of interest was done from the paraffin block. The areas of interest were de-waxed in three xylene washes before being re-hydrated in descending-graded ethanol. The selected samples were refixed in a glutaraldehyde and cacodylate buffer mixture, washed in cacodylate-sucrose buffer, and post-fixed in osmium tetroxide at 4 °C in a cold room. The samples were dehydrated in ascending graded ethanol prior to being impregnated in Epon 812 substitute (EMBed-812 Kit, Electron Microscopy Science, USA) at room temperature and polymerized at 60 °C for 48 h. Semithin sections were cut first from the resin block to confirm the presence of tissue in request. Then ultrathin sections were made using an Ultracut R ultramicrotome (Leica, Vienna, Austria). The mounted grids were examined at 80 kV under Jeol TEM Jem-1400 electron microscope (Jeol, Japan). We categorized each case according to TCs and/or Tps count detected in 3 examined TEM fields into: (0) none, (1) at least one was detected. (2) 2 and < 4 (3) more than 4.
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