Two liver cancer cell lines, HepG2 and PLC, were transfected with a FOXO1 inhibitor for 24 h. Then, the cells were treated with SAHA or NaB for 12 h and then immediately treated with 2% glutaraldehyde and 2% paraformaldehyde. The cells were fixed in sodium cacodylate buffer (pH 7.2) for 3 h at room temperature and then fixed at 4 ° C for 16 h. Postfixation was carried out for 1 h at room temperature in 1% osmium tetroxide in sodium dimethyl citrate buffer. After dehydration by a graded series of ethanol, all samples were kept in epoxy resin (Agar Scientific, AGR 1043) for 16 h prior to embedding. Ultrathin sections were collected on a Formvar-coated 200 mesh or single hole copper grid (Agar Scientific, AGS162) and stained with 2% uranyl acetate in 50% ethanol and lead citrate for 10 min each. Electron microscopic images were obtained using an HT7700 transmission electron microscope (Hitachi, Japan).
Ags162
Manufactured by Agar Scientific
Sourced in United Kingdom
The AGS162 is a laboratory device designed for general purpose use in scientific and research applications. It is a multi-functional instrument capable of performing various tasks within a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using ags162
1
Ultrastructural Analysis of FOXO1-Inhibited Liver Cancer Cells
2
Ultrastructural Analysis of Mycobacterial Infection in Zebrafish
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Mycobacteria in tail fin of 5 dpf zebrafish larvae as previously described (43 (link)). Wild type zebrafish larvae were infected with ~250 CFU M. marinum or M. avium at 2 dpf. At 3 dpi, the infected larvae were fixed in 2% glutaraldehyde and 2% paraformaldehyde in sodium cacodylate buffer (pH 7.2) for 3 h at room temperature after anesthetized properly. Subsequently, fixed samples were kept at 4°C for a further 16 h fixation. The next day, the samples were fixed in 1% osmium tetroxide in sodium cacodylate buffer (with 15mgr Potassium Ferrocyanide/ml) for 1 h at room temperature. All samples were kept in epoxy resin (Agar Scientific, AGR1043) for 16 h after the dehydration through a series of ethanol. Ultrathin sections were collected on Formvar coated 200 mesh or one hole copper grids (Agar Scientific, AGS162) stained with 2% uranyl acetate in 50% ethanol and lead citrate for 10 min each. The samples were imaged on a JEM- JEOL 1400 transmission electron microscope (Tokyo, Japan), which was equipped with an Olympus Megaview camera (Tokyo, Japan).
3
Zebrafish Larvae Ultrastructure Analysis
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Before being used for electron microscopy the zebrafish larvae were anesthetized with 200 µg/ml tricaine, imaged alive by CLSM and afterwards immediately fixated in 2% glutaraldehyde and 2% paraformaldehyde in sodium cacodylate buffer (pH 7.2) for 3 h at room temperature followed by fixation for 16 h at 4 °C. Postfixation was performed in 1% osmium tetroxide in sodium cacodylate buffer for 1 h at room temperature. After dehydration through a graded series of ethanol all specimens were kept in epoxy resin (Agar Scientific, AGR1043) for 16 h before embedding. Ultrathin sections were collected on Formvar coated 200 mesh or one hole copper grids (Agar Scientific, AGS162) stained with 2% uranyl acetate in 50% ethanol and lead citrate for 10 min each. Electron microscopy images were obtained with a JEOL JEM-1010 transmission electron microscope (Tokyo, Japan) equipped with an Olympus Megaview camera (Tokyo, Japan).
4
TEM Analysis of Chondrocytes in Larval Zebrafish
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Following treatment with DMSO or BafA1 for 3 h, larvae at 5 dpf were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) at 4°C, washed and then fixed in 0.2 M osmium in sodium cacodylate buffer with 1.5% ferrocyanide for 1 h at room temperature. After washing, larvae were placed into a sample processor for transmission electron microscopy (TEM) using a standard Epon resin protocol. Briefly, larvae were postfixed in reduced osmium, stained with uranyl acetate dehydrated in ethanol and infiltrated with Epon resin via propylene oxide, and polymerized at 60°C for 48 h.
Ultra‐thin sections (50 nm) of epon embedded larvae were cut using a diamond knife, collected on Formvar coated one‐hole copper grids (AGS162, Agar Scientific, UK) and observed using a Tecnai 12‐FEI 120 kV BioTwin Spirit transmission electron microscope. Images of chondrocytes from transverse sections of the ethmoid plate were collected, (n = 3 larvae per genotype, per condition) using an FEI Eagle 4k × 4k CCD camera and analyzed using the freehand selection tool and multi‐point counter in Fiji.60
Ultra‐thin sections (50 nm) of epon embedded larvae were cut using a diamond knife, collected on Formvar coated one‐hole copper grids (AGS162, Agar Scientific, UK) and observed using a Tecnai 12‐FEI 120 kV BioTwin Spirit transmission electron microscope. Images of chondrocytes from transverse sections of the ethmoid plate were collected, (n = 3 larvae per genotype, per condition) using an FEI Eagle 4k × 4k CCD camera and analyzed using the freehand selection tool and multi‐point counter in Fiji.
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