Cells were fixed with 2.5% glutaraldehyde (Sigma-Aldrich) in 0.1 M cacodylate buffer, pH 7.4, for 45 min at 4 °C, rinsed in buffer, postfixed in 1% OsO4 in 0.1 M cacodylate buffer, pH 7.4, dehydrated, and embedded in Epon resin (Agar Scientific). Grids were thoroughly rinsed in distilled water, stained with aqueous 2% uranyl acetate for 20 min. and photographed in a Zeiss EM 900 electron microscope. The number of autophagic vacuoles were counted under the Zeiss EM 900 electron microscope at ×12.000 magnification (48 μm2) for each treatment conditions. Autophagic vacuoles were classified as autophagosomes when met two or more of the following criteria: double membrane, compartments of 0.5 μm in diameter or larger, luminal uncompacted cytosolic material including organelles, absence of ribosomes attached to the cytosolic side of the membrane. We examined 70–100 fields per treatment condition and value are expressed as AVs per field. Finally, data were averaged to median values ± standard deviation (±SD) and used for statistical analysis.
Em900 electron microscope
Manufactured by Zeiss
Sourced in Germany
The EM900 is a transmission electron microscope (TEM) designed and manufactured by Zeiss. It is capable of magnifying and imaging samples at the nanoscale level. The EM900 utilizes an electron beam to illuminate the specimen and create a magnified image that can be observed on a viewing screen or captured digitally.
Automatically generated - may contain errors
Lab products found in correlation
Glutaraldehyde, by Electron Microscopy Sciences (5 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (4 mentions)
Jsm 6360 scanning microscope, by JEOL (3 mentions)
Em uc6 ultramicrotome, by Leica (3 mentions)
Zetasizer nano z, by Malvern Panalytical (3 mentions)
Formvar film, by Merck Group (2 mentions)
Em trim, by Leica (2 mentions)
Ultracut uct microtome, by Leica (2 mentions)
Anti nlrp3, by Cusabio (2 mentions)
Ultracut s ultramicrotome, by Leica (2 mentions)
Formaldehyde, by Serva Electrophoresis (2 mentions)
Glutaraldehyde, by Merck Group (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
Um ec7 ultramicrotome, by Leica (2 mentions)
Anti mmp 9, by Merck Group (1 mentions)
Anti mmp 2 primary antibody, by Novus Biologicals (1 mentions)
Em uc6, by Leica (1 mentions)
Glutaraldehyde, by Science Services (1 mentions)
Osmium tetroxide, by Electron Microscopy Sciences (1 mentions)
Uranyl acetate, by Agar Scientific (1 mentions)
73 protocols using em900 electron microscope
1
Quantifying Autophagic Vacuoles by Electron Microscopy
2
Ultrastructural Localization of MMP-2 and MMP-9
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For immunolocalization of MMP-2 and MMP-9, the promastigotes processing was performed as previously described [17 (link)]. The cells were fixed with 0.5% glutaraldehyde, 4% formaldehyde, 3.5% sucrose, 0.1 M sodium cacodylate and 5 mM sodium chloride. After that, the samples were dehydrated in an increasing series of ethanol at low temperature and embedded in LR White hydrophilic resin (Sigma Aldrich®). Ultrafine Sect. (60 μm) were obtained using an ultramicrotome (Leica EM UC6) and collected in gold grids. The grids were incubated with 50 mM NH4Cl solution, followed by incubation with PBS (pH 8.0) containing 1% BSA solution and 0.01% Tween 20, and then incubation with 10% goat serum. Subsequently, the samples were incubated with polyclonal anti-MMP-2 primary antibody (Novusbio®) or anti-MMP-9 (EMD Millipore®) at 1:100 overnight in a humid chamber at 4 °C, followed by incubation with goat anti-rabbit secondary antibody conjugated with colloidal gold 15 nm (Electron Microscopy Science©). Sample analysis was performed using an EM 900 Zeiss electron microscope.
3
Ultrastructural Analysis of Treated Parasites
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The ultrastructural study was carried out according to Brengio et al. (2000) . Briefly, treated or non-treated parasites were collected, centrifuged at 3000 rpm, and fixed with 2% glutaraldehyde in PBS (24 h at 4 ºC). Cells were washed twice with PBS, and then incubated overnight with 1% osmium tetroxide at 4 ºC. Parasites were washed twice and dehydrated with increasing concentrations of acetone, and pre-infiltrated in a 1:1 solution of acetone and Spurr Low Viscosity Kit, Ted Pella resin for 2 h. Cells were then centrifuged and embedded in pure Spurr Low Viscosity Kit, Ted Pella resin for 24 h at 60 ºC. Ultrathin sections were cut with a Power Tone XL ultramicrotome and contraststained with uranyl acetate and lead citrate. Samples were analyzed in an EM 900 Zeiss electron microscope.
4
Phage Visualization by Electron Microscopy
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A filtered high-titer phage lysate was centrifuged at 25,000×g for 60 min. The pellet was washed twice in ammonium acetate (0.1 M, pH 7.0). Phages were deposited on copper grids with carbon-coated Formvar films (Sigma-Aldrich Co., St. Louis, MO, USA) and stained for 10 s with uranyl acetate (2 %, pH 4.5). Excess liquid was blotted off and phages were examined using a Zeiss EM 900 electron microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) at the Laboratory of Microscopy Techniques, University of Wroclaw, Poland. The magnification was calibrated using T4 phage tail length (114 nm) as a standard.
5
Ultrastructural Analysis of Corpus Callosum
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As described above, mice were deeply anesthetized and transcardially perfused with PBS; half of the brain was dissected and immersion‐fixed in 4% PFA and stored at 4°C. Later, the brains were postfixed with 4% formaldehyde (Serva, Heidelberg, Germany), 2.5% glutaraldehyde (Science Services, Munich, Germany), and 0.5% NaCl in phosphate buffer pH 7.4 17 . Finally, the corpus callosum was postfixed with 2% OsO4 (Science Services) in 0.1 M phosphate buffer pH 7.3 and embedded in EPON (Serva) after dehydration with ethanol and propylene oxide. EPON blocks with embedded tissue were then trimmed, using a Leica EM TRIM (Leica, Vienna, Austria), to the size of the corpus callosum. In the following, ultrathin sections were stained with an aqueous solution of 4% uranyl acetate followed by lead citrate 18 . The pictures were taken in an unbiased random fashion with a Zeiss EM900 electron microscope (Zeiss, Oberkochen, Germany) using a side‐mounted 2k CCD camera (TRS, Waakirchen, Germany). Counting of myelinated and demyelinated axons was performed using NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA); a total area of 10*784 μm2 = 7840 μm2 was analyzed per mouse and every axon counted. The g ratio was calculated as the ratio between the number of demyelinated axons and the number of myelinated axons. Counting was performed on groups of n = 5 mice.
6
Mouse Organ Microscopy Protocol
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Methods for light- and electron-microscopy analysis of mouse organs were performed as described previously (Stinchi et al., 1999 (link)). Briefly, mice were deeply anaesthetized with an intraperitoneal injection of ketamine and xylazine. After pre-perfusion with 1% procaine in 0.1 M PBS, fixation was achieved with transcardial vascular perfusion with 6% glutaraldehyde in 0.1 M PBS. Tissue blocks were post-fixed with 2% osmium tetroxide and embedded in araldite. Semi-thin sections were stained with Toluidine Blue. Ultrathin sections were processed with uranyl acetate and lead citrate and viewed with a Zeiss EM 900 electron microscope (Zeiss).
7
Ultrastructural Visualization of Cellular Components
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For transmission electron microscopy (TEM), cells were processed as follows. Some cells were fixed with 2.5 % (v/v) glutaraldehyde and 2 % (v/v) PFA in 0.1 M phosphate buffer; pH 7.4, at 4 °C for 1 h, washed and incubated with 3,3’ diaminobenzidine (DAB, Sigma-Aldrich) (2 mg/ml in 50 mM Tris-HCl; pH 7.6) under simultaneous irradiation with two 8 W Osram Blacklite 350 lamps for 2 h at room temperature (spectral emission range between 430 and 470 nm, thus being suitable for FITC excitation). The cells were then post-fixed with 1 % osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) and 1.5 % potassium ferrocyanide (Sigma-Aldrich) at room temperature for 1 h, dehydrated with acetone and embedded in Epon (Agar Scientific, Assing, Monterotondo, Italy). Parallel samples, after aldehyde fixation, were incubated with DAB under light irradiation, dehydrated with ethanol and embedded in LR White resin. As negative controls, some samples were processed as described above but omitting both DAB incubation and exposure to the excitation light. Ultrathin sections were weakly stained with uranyl acetate (Agar Scientific). The samples were finally examined and photographed with a Zeiss EM 900 electron microscope at 80 KV (Carl Zeiss, Jena, Germany). The micrographs were then developed and digitalised [15 (link)].
8
Electron Microscopy Sample Preparation
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For scanning electron microscopy (SEM), samples were fixed in modified Karnovsky solution. Post-fixation was performed for 30 min in 1 % OsO4 in buffer solution (sodium cacodylate buffer at pH 7.4), followed by 15 min in 1 % tannic acid in buffer solution and additional 15 min in fresh solution of 1 % OsO4 at 4 °C. Then, specimens were decalcified as previously described for histological procedures and dehydrated in a graded ethanol series. Samples were critical point dried using CO2 as intermediate in a Balzers CPD 030 (Electron Microscopy Sciences, EUA), mounted on stubs, coated with gold in a Balzers SCD 050 sputter coater (Electron Microscopy Sciences, EUA), and observed in a Zeiss DSM 940 (Carl Zeiss, Oberkochen, Germany). For transmission electron microscopy (TEM), post-fixation was performed for 1 h in 1 % OsO4 in buffer solution. Larval samples were embedded in Epoxy resin; ultrathin sections (50–70 nm) were cut using a Leica Ultracut UCT microtome (Leica, Illinois, USA), mounted on copper slot-grids, contrasted with uranyl acetate and lead citrate, and analyzed using a Zeiss EM 900 electron microscope (Carl Zeiss, Oberkochen, Germany).
9
Imaging NLRP3 in Neutrophils
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WT and AnxA1-/- nigericin-stimulated neutrophils were processed for transmission electron microscopy analysis as previously described [13 (link)]. To detect NLRP3, ultrathin sections (~90 nm) from neutrophils were submitted for immunocytochemistry, using a rabbit polyclonal antibody anti-NLRP3 1:200 (Cusabio, Houston, TX, USA) followed by a goat anti-rabbit IgG antibody (1:50) conjugated to 15 nm colloidal gold (British Biocell, Cardiff, UK). Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a ZEISS EM900 electron microscope (Carl Zeiss, Oberkochen, Germany).
10
Imaging of AnxA1 and NLRP3 in Macrophages
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WT and AnxA1-/- nigericin-stimulated macrophages were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde, 0.1% sodium cacodylate buffer (pH 7.4) for 24 h at 4 °C. Samples were washed in sodium cacodylate, dehydrated through a graded methanol series, and embedded in LR Gold (Sigma Aldrich Corp., St. Louis, MO, USA).
To detect AnxA1 and NLRP3, ultrathin macrophage sections (~90 nm) were submitted for immunocytochemistry, as previously described [19 (link)]. To detect the proteins, the sheep polyclonal antibody anti-AnxA1 (1:200) and rabbit polyclonal antibody anti-NLRP3 (1:200; Cusabio, Houston, TX, USA), following a donkey anti-sheep IgG and goat anti-rabbit IgG antibody (1:50) conjugated to 10-nm and 20-nm colloidal gold (British Biocell, Cardiff, UK), respectively, were used. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a ZEISS EM900 electron microscope (Carl Zeiss, Jena, Germany). Randomly photographed sections of macrophages were analyzed using Axiovision software. The density of immunogold (number of gold particles/µm2) was calculated and reported as the mean ± SEM of 20–40 cells per experimental condition.
To detect AnxA1 and NLRP3, ultrathin macrophage sections (~90 nm) were submitted for immunocytochemistry, as previously described [19 (link)]. To detect the proteins, the sheep polyclonal antibody anti-AnxA1 (1:200) and rabbit polyclonal antibody anti-NLRP3 (1:200; Cusabio, Houston, TX, USA), following a donkey anti-sheep IgG and goat anti-rabbit IgG antibody (1:50) conjugated to 10-nm and 20-nm colloidal gold (British Biocell, Cardiff, UK), respectively, were used. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a ZEISS EM900 electron microscope (Carl Zeiss, Jena, Germany). Randomly photographed sections of macrophages were analyzed using Axiovision software. The density of immunogold (number of gold particles/µm2) was calculated and reported as the mean ± SEM of 20–40 cells per experimental condition.
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