For TEM analysis, samples were fixed in Karnovsky’s Fixative consisting of 8% PFA, 25% glutaraldehyde (Agar Scientific, Stansted, UK), and 0.2 M cacodylate buffer (Agar Scientific) for 1 hour before washing in 0.1 M cacodylate buffer. Samples were further fixed in 1% osmium tetroxide (Agar Scientific) for 1 hour, washed in 0.1 M cacodylate buffer before being dehydrated through an ethanol series before embedding in Epon resin (Agar Scientific) containing dodecenylsuccinic anhydride (DDSA), methyl nadic anhydride (MNA) and benzyldimethylamine (BDMA). Ultra-thin (70 nm) sections were obtained using a diamond knife (Agar Scientific) and loaded onto formvar-coated slot grids (Agar Scientific). Grids were then stained with uranyl acetate (Agar Scientific) and lead citrate (Sigma Aldrich) prior to imaging using a Hitachi H7600 electron microscope.
Diamond knife
Manufactured by Agar Scientific
The Diamond knife is a specialized laboratory tool designed for ultra-thin sectioning of samples for electron microscopy. It features a monocrystalline diamond edge that provides an exceptionally sharp and durable cutting surface, enabling the preparation of high-quality, ultra-thin sections with minimal compression and distortion.
Automatically generated - may contain errors
Lab products found in correlation
Glutaraldehyde, by Agar Scientific (1 mentions)
Lead citrate, by Merck Group (1 mentions)
Uranyl acetate, by Agar Scientific (1 mentions)
Osmium tetroxide, by Agar Scientific (1 mentions)
0.1 m cacodylate buffer, by Agar Scientific (1 mentions)
Epon resin, by Agar Scientific (1 mentions)
Formvar coated slot grids, by Agar Scientific (1 mentions)
H 7600 electron microscope, by Hitachi (1 mentions)
2 protocols using diamond knife
1
Transmission Electron Microscopy Sample Preparation
2
Ultrastructural Analysis of SH-SY5Y Cells
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SH-SY5Y cells were seeded at a density of 1.2 × 10 5 /ml in 100 mm dishes and were allowed to attach for 24 h. Following treatment with rutin and MPP + as described in section 2.2.1, cells were centrifuged in ice-cold PBS at 3000 rpm for 5 min, fixed in 500 μL of 2.5% phosphate-buffered glutaraldehyde and post-fixed in 1% osmium tetroxide in the same buffer. The fixed samples were subsequently processed for transmission electron microscopy. A Reichert ultramicrotome (SMM Instruments, Johannesburg, South Africa) with a diamond knife (Agar Scientific, Randburg, South Africa) was used to make silver to gold sections. Thin sections on copper grids were examined using a Jeol JEM 1011 transmission electron microscope at 80 kV (Advanced Laboratory Solutions, Johannesburg, South Africa). Electron micrographs were produced with a Mega View III digital camera fitted onto the microscope and by means of the ITEM software package (Advanced Laboratory Solutions, Johannesburg, South Africa).
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