Megaview 2 camera

PKH26 stained HeLa cells on coverslips were fixed with 2.5% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4° C for 1 h, washed and incubated with 3,3’ diaminobenzidine (DAB) (20 mg/10 mL in 0.05 M Tris HCl, pH 7.6) under simultaneous irradiation with two 8W Osram Blacklite 350 lamps for 2 h at room temperature. These lamps have two emission peaks of high intensity at 550 and 580 nm, thus being compatible with the excitation maximum (551 nm) of PKH26 dye. The cells were then post-fixed with 1% OsO₄ at room temperature for 1 h, dehydrated with acetone and embedded in Epon. As controls, some PKH26 stained cells were processed as described above but omitting either DAB incubation or exposure to light; in addition, unstained cells were exposed to DAB incubation and/or exposure to light.
Thin sections were weakly stained with either 2.5% aqueous solution of uranyl acetate for 1-2 min or 2.5% to 5% aqueous solution of gadolinium triacetate for 10 min (gadolinium triacetate is a good substitute for uranyl acetate to enhance contrast of osmicated samples^{30 (link)}). The sections were observed in a Philips Morgagni transmission electron microscope (FEI Company) operating at 80kV and equipped with an Olympus Megaview II camera for digital image acquisition.