Infected monolayers and ileum fragments were first fixed in 2% glutaraldehyde (EMS, USA) for at least 24 h at 4°C. After primary fixation, cells and fragments were washed 3 times with PBS (10 min) and subjected to secondary fixation with 1% osmium tetroxide (EMS, USA) in 0.1 M sodium cacodylate buffer for 30 min. After being washed three times with distilled water, preparations were dehydrated through a graded ethanol series (50%, 75%, 85%, 95% and 100%), and propylene oxide (100%). Preparations were then gradually embedded in Araldite, which was allowed to polymerize for 24–48 h at 60°C. Ultrathin sections were placed on Formvar (EMS, USA) coated 200 mesh copper grids and stained with 4% aqueous uranyl acetate (Merck, Germany) and Reynold's lead citrate (Merck, Germany). Grids were examined under TEM (LEO 906E– Zeiss, Germany) at 80 kV [48 (link)].
Reynold s lead citrate
Manufactured by Merck Group
Sourced in Germany
Reynold's lead citrate is a chemical compound used in electron microscopy as a staining agent for the contrast enhancement of biological specimens. It is a heavy metal salt that binds to the negatively charged sites within the sample, increasing the electron density and improving the visualization of cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Uranyl acetate, by Merck Group (10 mentions)
Potassium ferrocyanide, by Merck Group (5 mentions)
Epon resin, by Merck Group (5 mentions)
Temcam f416, by TVIPS (4 mentions)
Aqueous uranyl acetate, by Merck Group (3 mentions)
Em906, by Zeiss (3 mentions)
Acetone, by Merck Group (3 mentions)
1010 transmission electron microscope, by JEOL (3 mentions)
Leo 906e, by Zeiss (2 mentions)
Temcam f416 digital camera, by TVIPS (2 mentions)
Eclipse ti s inverted microscope, by Nikon (2 mentions)
Ultracut microtome, by Merck Group (2 mentions)
Cm100, by TVIPS (2 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Osmium tetroxide, by Merck Group (1 mentions)
Quanta 250, by Thermo Fisher Scientific (1 mentions)
Ultracut s ultramicrotome, by Leica (1 mentions)
0.1 m sodium cacodylate buffer, by Serva Electrophoresis (1 mentions)
Agarose, by Merck Group (1 mentions)
Glutaraldehyde, by Serva Electrophoresis (1 mentions)
19 protocols using reynold s lead citrate
1
Ultrastructural Examination of Infected Cells
2
TEM Imaging of 3D Bioreactor Cells
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For TEM, part of the 3D bioreactor cell compartment was fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (both from Serva) over night, followed by postfixing in 1% OsO4 (Science Services) with 0.8% potassium ferrocyanide (Merck) in 0.1 M sodium cacodylate buffer for 1.5 h. Subsequently, samples were progressively dehydrated in ethanol and then embedded in Epon (Serva). Ultrathin sections were stained with uranyl acetate and Reynold's lead citrate (Merck) and microphotographs were taken using an electron microscope EM 906 (Carl Zeiss).
3
TEM Analysis of Bovine PMN-T. gondii Interaction
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For TEM analysis, 1 x 107 bovine PMN were confronted with vital 2 x 107T. gondii tachyzoites, and after 2 h of co-incubation, cells were fixed in glutaraldehyde (2.5% final concentration, Merck). The cells were centrifuged at 1000 × g for 10 min and the pellet was stored at 4 °C until further use according to (7 (link)) and (29 (link)). Briefly, the cell pellet was washed and post-fixed in buffer containing 1% osmium tetroxide (Merck). After thoroughly washing in distilled water, the samples were incubated overnight in 2% aqueous uranyl acetate (Merck) at 4 °C, dehydrated in ethanol and embedded in Epon (all Merck). Ultrathin sections of the cured blocks were mounted on formvar-coated grids and stained with uranyl acetate and Reynolds lead citrate (both Merck). The sections were inspected in a ZEISS EM912 AB microscope (Oberkochem, Germany) at the Institute of Anatomy and Cell Biology of the Justus Liebig University Giessen, Germany.
4
Ultrastructural Analysis of 3D Cell Cultures
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The 3D co-cultures were fixed with 3% glutaraldehyde in 100 mM sodium cacodylate buffer, pH 7.4 for 45 min, postfixed in 1% OsO4 for 50 min, and washed with cacodylate buffer. After embedding in 1% agar blocks, the samples were dehydrated in increasing ethanol series (50, 70, 96, and 100%), treated with 100% acetone, and embedded in Durcupan resin (Merck). Ultrathin sections were prepared using LKB 8802A Ultramicrotome, stained with uranyl acetate and Reynold’s lead citrate (Merck), and examined with FEI Morgagni 286(D) transmission electron microscope. The cells in the schematics were segmented manually.
5
Transmission and Scanning Electron Microscopy Protocol
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Tissue fragments obtained from in vivo experiments were primarily fixed in 2% glutaraldehyde (EMS, USA) and post-fixed with 1% osmium tetroxide (EMS) prepared with 0.1 M sodium cacodylate buffer. After fixation, specimens were dehydrated through a graded series of ethanol solutions (50%, 75%, 85%, 95%, and 100%) and propylene oxide (100%), and gradually embedded in Araldite resin. Ultrathin sections were placed onto 200 mesh copper grids previously coated with Formvar (EMS), and stained with 2% aqueous solution of uranyl acetate (Merck) and Reynold’s lead citrate (Merck). Grids were then examined under TEM (LEO 906E – Zeiss, Germany) [16 (link)].
For Scanning Electron Microscopy (SEM) analyses, tissue fragments were similarly fixed, post-fixed, and dehydrated as described for TEM. After dehydration with 100% ethanol, specimens were submitted to a critical point dry with carbon dioxide, and mounted onto SEM stubs. After receiving a thin layer of gold, specimens were examined under SEM (QUANTA 250 - FEI Company, Netherlands) at 12.5 kV [16 (link)].
For Scanning Electron Microscopy (SEM) analyses, tissue fragments were similarly fixed, post-fixed, and dehydrated as described for TEM. After dehydration with 100% ethanol, specimens were submitted to a critical point dry with carbon dioxide, and mounted onto SEM stubs. After receiving a thin layer of gold, specimens were examined under SEM (QUANTA 250 - FEI Company, Netherlands) at 12.5 kV [16 (link)].
6
Ultrastructural Analysis of Mitotane-Treated Cells
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For transmission electron microscopy, two resistant and two nonresistant clones were thawed and cultured to confluence without mitotane. Cells were seeded on a 6-well plate (2 × 106 cells per well). After 24 h, cells were treated with 50 µM mitotane or vehicle control (DMSO). After 72 h, cells were washed once with ice cold PBS and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (both from Serva, Heidelberg, Germany) at room temperature for 30 min. Afterwards, fixation buffer was changed, and cells were stored at 4°C for 2–14 days. Cells were postfixed in 1% OsO4 (Science Services, Munich, Germany) and 0.8% potassium ferrocyanide (Merck) in 0.1 M sodium cacodylate buffer for 1.5 h and then progressively dehydrated in ethanol, followed by embedding in Epon (Serva). Ultrathin sections (70 nm) were prepared using an Ultracut S Ultramicrotome (Leica), stained with uranyl acetate and Reynold’s lead citrate (Merck), and microphotographs were taken using an electron microscope EM906 (Carl Zeiss).
7
Ultrastructural Analysis of T Cell Mitochondria
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Naïve and polyclonally activated CD8 + T cells were fixed with 0.1 M sodium cacodylate buffer (Serva) supplemented with 2.5 % glutaraldehyde (Serva) for 30 min at room temperature, stored by 4 °C and submitted to the Core Facility for Electron Microscopy (Charité -Universitätsmedizin Berlin). Samples were postfixed with 0.1 M sodium cacodylate buffer supplemented with 1 % osmium tetroxide (Science Services) and potassium ferrocyanide (Merck) and embedded in 1 % agarose (Sigma-Aldrich). Embedded samples were dehydrated in a graded ethanol (Merck) series and embedded in epon (Serva). Ultrathin sections of 70 nm were prepared on a Leica Ultracut S (Leica Biosystems). Sections were stained with 4 % uranyl acetate (Serva) and Reynold's lead citrate (Merck). Micrographs were taken with a Zeiss EM 906 transmission electron microscope at 80 kV acceleration (Carl Zeiss) and a slow scan 2k CCD camera (TRS). Mitochondria and cristae were identified manually and the measures were outlined with an optical pen using ImageJ v1.53a software. The roundness factor was calculated as 4*area/pi*sqr(major axis) and a maximal distance of 75 nm between mitochondria and ER was defined as MERC.
8
Electron Microscopy Sample Preparation
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After NP exposure (as described above), cells were rinsed with PBS, fixed in 2.5% glutaraldehyde 2% PFA in cacodylate buffer (0.1 M pH 7.4) for 1h30 at room temperature, then rinsed with cacodylate buffer. Post-fixation was achieved in 1% osmium tetroxide 1.5 % potassium ferrocyanide solution at room temperature for one hour. After extensive washings with water, pellets were included in 2% LMP agarose and cut in mm 3 blocks. They were dehydrated through a graded series of ethanol before being embedded in Epon resin. Ultra-thin sections of 70 nm were cut with an EM UC6 ultramicrotome (Leica Microsystems) and stained for 10 min with 2% uranyl acetate (Merck) and 3 min with Reynolds lead citrate (Agar). They were then observed in TEM with a JEOL JEM-1400 electron microscope operating at 120 kV.
9
Transmission Electron Microscopy for ER Quantification
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Sorted cells were fixed in 2.5% glutaraldehyde solution (EMS) 1h at room temperature, and directly postfixed with 1% osmium tetroxide (EMS)/1.5% potassium ferrocyanide (Sigma) for 1h at room temperature. After several washes and dehydration in acetone (Sigma), cells were then embedded in Epon resin (Sigma). Sections of 50 nm were prepared on a Leica Ultracut microtome (Leica Mikrosysteme), followed by poststaining with 4% uranyl acetate (Sigma) and Reynolds’ lead citrate (Sigma). Images were recorded with a transmission electron microscope Philips CM100 (ThermoFisher Scientific) at an acceleration voltage of 80 kV with a TemCam-F416 digital camera (TVIPS). Analysis and quantification were performed by using ImageJ software. For assessing ER extension, each dot represents the total length of ER compartment assessed by analyzing the length of each selected line of ER in the ROI manager.
10
Transmission Electron Microscopy for ER Quantification
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