Cm100 transmission electron microscope

HUVECs were fixed with 1% glutaraldehyde in phosphate buffer, pH 7.2. The cells were washed and scraped off the plates in phosphate buffer and pelleted. The pellet was postfixed with 1% OsO₄ in phosphate buffer for 1 h at 4 °C and then dehydrated in a graded series of ethanol and embedded in Epon according to routine techniques (Luft 1961). Ultrathin sections were obtained using a Reichert Ultracut ultramicrotome and mounted on naked nickel grids. Sections were stained with 3% aqueous uranyl acetate and lead citrate, and examination was performed with a Philips CM100 transmission electron microscope. Electron micrographs were captured using an AMT XR80 digital camera.

HT22 or N2a cells were seeded in 100 mm dish and cultured in DMEM media with 10% FBS and 50 units of PSN mixture as supplements. After reaching a confluence between 60 and 70%, HT22 or N2a cells were treated with drugs including Cory-B (20 µM), Torin1 (250 nM), Fe65-EXO, Fe65-EXO-Cory-B, or Fe65-EXO-Curcumin (10 µM) for a period of 24 h. After treatment, the medium was discarded, and cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h at room temperature. The fixed cells were collected via scraping from the cell culture plate into a centrifuge tube and then centrifuged. The pellet of the fixed cells was dissolved and rinsed in 0.1 M sucrose in cacodylate buffer and post-fixed with 1% osmium tetroxide for dehydration. The dehydrated cells were embedded in Poly/Bed® 812 for ultra-thin slicing to examine their components, followed by sectioning using an ultramicrotome with thickness around 100 to 200 nm before being fixed in carbon-coated electron microscopy grids and viewed under a Philips CM100 transmission electron microscope at the QMH Electron Microscope Unit, Queen Mary Hospital, University of Hong Kong, Hong Kong.
For the examination of synapse formation in the mice brain, the tissue samples were fixed, dehydrated, embedded, sectioned, stained with uranyl acetate, followed by mounting on the grid for electron microscopy examination.

The procedure for gold nanoparticles (gold NPs) preparation was adopted from Hayat (Hayat 1989). Gold NPs were prepared by reduction of HAuCl₄ with sodium citrate. The starting solution of 106 mL of 2.2 mM (0.064%, w/v) sodium citrate was brought to a boil and 1 mL of 24.3 mM HAuCl₄ (0.955%, w/v) was added with rapid mixing. The reaction was completed within 1–2 min and the solution was further boiled for 15 min. Gold NPs in solution were centrifuged at 6500× g for 30 min at 4 °C. Nine tenths of the supernatant was discarded to increase the concentration of gold NPs in the solution. Next, 5 μL of the gold NPs solution were deposited onto copper grids and air-dried. Images of gold NPs were taken at 20,000–100,000× magnifications with a Philips CM100 transmission electron microscope (Philips, Tokyo, Japan) at 80 kV using an AMT camera (Advanced Microscopy Techniques Corp., Woburn, MA, USA).

Keratinocytes treated with TET for 2, 6, and 24 h and untreated cells, used as control, were grown to confluent monolayers on 10 cm Petri dishes. The cells were fixed in 2.5% glutaraldehyde in cacodylate buffer for 3 h at 4 °C and then post-fixed in 1% osmium tetroxide for 2 h at 4 °C, dehydrated in a graded series of alcohol, embedded in Araldite resins, and polymerised in the oven for 48 h at 60 °C. Ultrathin sections of 60 nm were cut with an ultramicrotome (Ultratome Reichert SuperNova Leica, Wien, Austria), stained with uranyl acetate and lead citrate, and examined in a Philips CM100 transmission electron microscope [44 (link)].

Flies were prepared for transmission electron microscopy essentially as described in Longley and Ready (1995) (link) with modifications. Ultrathin sections were cut with a diamond knife using a Reichert-Jung Ultracut microtome. Sections were stained with Reynold's lead citrate and 2% aqueous uranyl acetate and were observed with a Philips CM-100 transmission electron microscope. For electron tomography, 420 nm thick sections were cut and a 200 kV Tecnai G2 20 electron microscope (FEI) equipped with a Gatan CCD camera was used to record serial tilts of ±60° in increments of 1°. Tomogram reconstruction was carried out using IMOD software (Kremer et al., 1996 (link)).