Another two washes with buffer were performed before uranyl acetate
staining (1 % uranyl acetate
in 10 % ethanol for 8 hours).
Ultrathin sections were taken with LKB ultramicrotome III and stained with uranyl acetate
and lead citrate.
Samples were stained en bloc within a range of 1/5 stock RR solution and 2% uranyl acetate
(22), then dehydrated in a ascending alcohol series and embedded in epon 812.
Reagents and poison: Electron microscopy reagents (EPON EMBed-812 Kit, uranyl acetate
, osmium tetroxide, lead citrate and glutaraldehyde) were purchased from Electron Microscopy Sciences (Hatfield, PA, USA).
Post-stain procedures for 100-nm sections observed with electron microscopy varied, with the sample being treated either with (1) 2% aqueous uranyl acetate
(15 min) and 0.5% aqueous lead citrate (1 min) with 3 washes of 20 s with DI water in between; (2) only 2% aqueous uranyl acetate
(15 min); or (3) only 0.5% aqueous lead citrate (1 min).
(e) Ultrastructural examination shows osmophilic globular hemoglobin casts in tubular lumen (uranyl acetate
and lead citrate fixation, 10,000x).
For visualization using TEM, uranyl acetate
was used as a negative staining reagent for the liposomes .
Poly(vinyl alcohol) (PVA, Mw of 15kDa), uranyl acetate
dehydrate (98%), dimethyl sulfoxide (DMSO, 99.5%), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), fluorescein isothiocyanate (FITC), and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoli-um bromide (MTT) were supplied by Sigma-Aldrich (St.
Sections were stained with uranyl acetate
and lead citrate, and examined.
(a) H&E stain, x40; (b) GiemsaOrcein stain, x100; (c) and (d) uranyl acetate
& lead citrate, original magnification x 13.000.
After uranyl acetate
staining, the pellet was rinsed with 1.5 ml of distilled H2O immediately and was then repelleted by centrifugation.
12 [micro]l of the dilutions were allowed to adsorb on carbon coated copper grid for 1 min followed by negative staining with aqueous uranyl acetate