Tissue samples were fixed for 72 h in a mixture of 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Samples were then washed in 0.1 M cacodylate buffer and postfixed with 1% osmiumtetroxide (OsO4)/1.5% potassiumferrocyanide (KFeCN6) for 1 h at room temperature. After washes in water and 50 mM maleate buffer pH 5.15 (MB), the samples were incubated in 1% uranyl acetate in MB for 1 h, washed in MB and water, and dehydrated in grades of alcohol (10 min each: 50%, 70%, 90%, 2 × 10 min 100%). The tissue samples were then put in propyleneoxide for 1 h and infiltrated overnight in a 1:1 mixture of propyleneoxide and TAAB Epon. The following day the samples were embedded in fresh TAAB Epon and polymerized at 60°C for 48 h. Semi-thin (0.5 μm) and ultrathin sections (50–80 nm) were cut on a Reichert Ultracut-S microtome (Leica). Semi-thin sections were picked up on glass slides and stained with toluidine blue for examination by light microscopy to find affected areas in the tissue. Ultrathin sections from those areas were picked up onto formvar/carbon coated copper grids, stained with 0.2% lead citrate and examined in a JEOL 1200EX transmission electron microscope (JOEL, Akishima, Tokyo, Japan). Images were recorded with an AMT 2k CCD camera.
Reichert ultracut s microtome
Manufactured by Leica
Sourced in Germany
The Reichert Ultracut S microtome is a precision instrument designed for the preparation of thin sections of materials for microscopic examination. It is capable of producing sections ranging from 0.05 to 2 micrometers in thickness, allowing for the detailed analysis of a wide variety of samples.
Automatically generated - may contain errors
Lab products found in correlation
Morada digital camera, by Olympus (4 mentions)
Je 1400, by JEOL (4 mentions)
1200 ex transmission electron microscope, by JEOL (3 mentions)
Tecnai g2 spirit biotwin, by Thermo Fisher Scientific (2 mentions)
Cm 120 transmission electron microscope, by Thermo Fisher Scientific (2 mentions)
Ultra diamond knife, by Electron Microscopy Sciences (2 mentions)
Ultrostainer, by Leica (2 mentions)
Diamond knife, by Leica (1 mentions)
1010 transition electron microscope, by JEOL (1 mentions)
Jem 1230 transmission electron microscope, by JEOL (1 mentions)
Formvar coated copper grid, by Electron Microscopy Sciences (1 mentions)
Ethanol, by Merck Group (1 mentions)
Uranyl acetate, by Electron Microscopy Sciences (1 mentions)
Osmium tetroxide, by Electron Microscopy Sciences (1 mentions)
Embed 812 embedding kit, by Electron Microscopy Sciences (1 mentions)
Aclar, by Ted Pella (1 mentions)
Amt 2k ccd camera, by Advanced Microscopy Techniques (1 mentions)
Amt 2k ccd camera, by JEOL (1 mentions)
Em pact2, by Leica (1 mentions)
Lowicryl hm20, by Polysciences (1 mentions)
27 protocols using reichert ultracut s microtome
1
Ultrastructural Analysis of Tissue Samples
2
Ultrastructural Bone Tissue Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were fixed in 2.5% Glutaraldehyde overnight followed by 2% EDTA for decalcification overnight. The first step was 100% ethanol dehydration of the samples and then infiltration procedure of 1-h Agar 100 epoxy resin mix, 1-h propylene oxide and 4 h pure epoxy resin. Medium sized bone nodules were chosen and cut trans sectionally for ~ 70 nm (nm) sections through a Reichert ultra-cut S microtome with a diamond knife (Leica, Milton Keynes, UK). A JEOL 1010 transition electron microscope (TEM; Tokyo, Japan), operated at 120 kV, was used for imaging of the section.
3
Ultrastructural Analysis of T Cell Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For T cell stimulation, 2 × 106/ml Jurkat T cells were incubated with 10 µg/ml biotin-conjugated anti–human CD3ε antibody (BioLegend), followed by cross-linking with 20 µg/ml streptavidin (Cell Signaling Technology) for 10 min at 37°C. Stimulation was stopped by adding the same volume of 2× fixative (2.5% glutaraldehyde, 1.25% paraformaldehyde, and 0.03% picric acid in 0.1 M sodium cacodylate buffer, pH 7.4). Cells were spun down at 1,500 rpm for 5 min. The cell pellet was fixed for 2 h at room temperature in the fixative, washed in 0.1 M cacodylate buffer, and postfixed with 1% osmium tetroxide (OsO4)/1.5% potassium ferrocyanide (KFeCN6) for 1 h, washed in water, and incubated in 1% aqueous uranyl acetate for 1 h. Cells were subsequently dehydrated in grades of alcohol (10 min each; 50%, 70%, 90%, 2 × 10 min 100%). The samples were then put in propylene oxide for 1 h and infiltrated overnight in a 1:1 mixture of propylene oxide and TAAB epon (Marivac Canada Inc.). The next day, the samples were embedded in TAAB epon and polymerized at 60°C for 48 h. Ultrathin sections (60 nm) were cut on a Reichert Ultracut-S microtome (Leica), transferred onto copper grids, stained with lead citrate, and examined in a JEOL 1200EX transmission electron microscope (JEOL) or a Tecnai G2 Spirit BioTWIN (FEI). Images were recorded with an AMT 2k CCD camera.
4
Freeze Substitution and Embedding for TEM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gravid adults were frozen substituted in acetone (J.T. Baker, Phillipsburg, NJ, USA) containing 2% osmium tetroxide (Electron Microscopy Science, Hatfield, PA, USA) and 0.2% uranyl acetate (Electron Microscopy Science) in a Leica EM automatic freeze substitution machine (Leica). The temperature was raised from −90 °C to room temperature for 22 h (5 °C/h). The samples were dehydrated in 100% ethanol (Sigma-Aldrich) for 24 h, followed by two washes. The washed samples were embedded in the EMBed-812 embedding kit (Electron Microscopy Science) and incubated at 70 °C for 48 h. The 70 nm thin sections were cut by using a Reichert ultracut S microtome (Leica). Thin sections were collected on Formvar-coated copper grids (Electron Microscopy Science) and post-stained with 0.8% uranyl acetate (Electron Microscopy Science) in 40% methanol (J.T.Baker) followed by aqueous lead citrate (Electron Microscopy Science). Thin sections were viewed in a JEM-1230 transmission electron microscope (JEOL, Akishima, Tokyo, Japan).
5
Ultrastructural Analysis of T. cruzi-Infected NHDF Cells
6
Adenovirus Infection of Keratocytes
7
High-Resolution Cellular Ultrastructure Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For EM analysis, cells were high pressure frozen, freeze-substituted, sectioned, and stained as described previously (Giddings et al., 2001 (link)). First, vacuum filtration was performed to collect cells onto a 0.45 µm polycarbonate filter (EMD Millipore). Cells were transferred and cryo-immobilized by using the EM PACT2 (Leica), a high-pressure freezing machine operating at a pressure of ∼2,045 bar. For freeze-substitution, an EM-AFS2 device (Leica) together with a freeze-substitution solution of 0.1% glutaraldehyde, 0.2% uranyl acetate, and 1% water dissolved in anhydrous acetone was used, followed by stepwise infiltration in Lowicryl HM20 (Polysciences, Inc.), starting at a low temperature of −90°C. The samples were then exposed to UV light at −45°C for 48 h and gradually warmed up to 20°C to allow polymerization. Serial sectioning of embedded cells, to generate 60–70-nm-thick sections, was done using a Reichert Ultracut S Microtome (Leica). Sections were poststained with uranyl acetate and lead citrate. Finally, images were captured with an electron microscope (CM120; Philips Electronics) operated at 120 kV.
8
Tissue Ultrastructure Examination Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples were fixed for 72 h in a mixture of 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Samples were then washed in 0.1 M cacodylate buffer and postfixed with 1% osmiumtetroxide (OsO4)/1.5% potassiumferrocyanide (KFeCN6) for 1 h at room temperature. After washes in water and 50 mM maleate buffer pH 5.15 (MB), the samples were incubated in 1% uranyl acetate in MB for 1 h, washed in MB and water, and dehydrated in grades of alcohol (10 min each: 50%, 70%, 90%, 2×10 min 100%). The tissue samples were then put in propyleneoxide for 1 h and infiltrated overnight in a 1:1 mixture of propyleneoxide and TAAB Epon. The following day the samples were embedded in fresh TAAB Epon and polymerized at 60°C for 48 h. Semi-thin (0.5 μm) and ultrathin sections (50–80 nm) were cut on a Reichert Ultracut-S microtome (Leica). Semi-thin sections were picked up on glass slides and stained with toluidine blue for examination by light microscopy to find affected areas in the tissue. Ultrathin sections from those areas were picked up onto formvar/carbon coated copper grids, stained with 0.2% lead citrate and examined in a JEOL 1200EX transmission electron microscope (JOEL, Akishima, Tokyo, Japan). Images were recorded with an AMT 2k CCD camera.
9
Ultrastructural Analysis of HAdV-D37 Infection in siRNA-treated HEK293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 siRNA treated cells were infected with HAdV-D37 at an MOI of 0.1 for 72 hrs, and then fixed in 2.5% glutaraldehyde, 1.25% paraformaldehyde, and 0.03% picric acid in 0.1M cacodylate buffer for 1 hr and sent for transmission electron microscopy (https://electron-microscopy.hms.harvard.edu/ ). The samples were post-fixed in 1% osmium tetroxide (OsO4)/1.5% potassium ferrocyanide (KFeCN6) for 30 min, washed and incubated in 1% aqueous uranyl acetate for 30 min followed by two washes and subsequent dehydration in graded alcohol (5 min each, 50%, 70%, 95%, 2x 100%). The samples were then removed from the dish in propylene oxide and incubated overnight in a 1:1 mixture of propylene oxide and TAAB Epon (TAAB Laboratories Equipment Ltd, Aldermaston, Berks). The next day samples were embedded in fresh TAAB Epon and polymerized at 60° C for 48 hrs. Ultrathin sections (~60 nm) cut on a Reichert Ultracut-S microtome (Leica, Buffalo Grove, IL), placed on copper grids, stained with lead citrate, and examined in a JEOL 1200EX transmission electron microscope (JEOL Inc. Peabody, MA). Images were recorded with an AMT 2k CCD camera (AMT, Woburn, MA).
10
Ultrastructural Analysis of Plant Leaves
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Small pieces of leaves were cut and fixed in 3% Glutaraldeyde in 0.1M Cacodylate buffer (pH 7.4) 10 hours at room temperature in a desiccator and then transferred to 40°C for the continuation of fixation overnight. The tissues were washed in cacodylate buffer and postfixed and stained with 2% osmium tetroxide, 1.5% potassium ferricyanide in 0.1M cacodylate buffer for 2 hours. Tissues were then washed in cacodylate buffer and dehydrated through a graded series of ethanol treatments for 10 min each step, followed by 100% anhydrous ethanol 3 times, 20 min each, and propylene oxide 2 times, 10 min each. The tissues were infiltrated with increasing concentrations of Agar 100 resin in propylene oxide for 16 hours each step. The tissues were embedded in fresh resin in a 600°C oven for 48 hours. Ultrathin sections, approximately 80 nm thick, were cut on a Leica Reichert Ultracut S microtome, collected onto 200 Mesh carbon-formvar coated copper grids, and stained with Uranyl acetate followed by Reynold's Lead Citrate for 10 min. The sections were examined using Tecnai 12 TEM 100kV (Phillips, Eindhoven, the Netherlands) equipped with MegaView II CCD camera and Analysis® version 3.0 software (SoftImaging System GmbH, Münstar, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!