Sis megaview 3 plate camera

Transmission electron microscopy (TEM) was used to evaluate the ultrastructure of RPE monolayers. Briefly, 3–10 month old RPE cultures grown on polyester transwell membranes or on electrospun scaffolds were fixed with primary fixative comprising 3% glutaraldehyde, 4% formaldehyde in 0.1 M PIPES buffer (pH 7.2) for a minimum of 1 h. Specimens were then rinsed in 0.1 M PIPES buffer, post-fixed in 1% buffered osmium tetroxide (1 h), rinsed in buffer, block stained in 2% aqueous uranyl acetate (20 min), dehydrated in an ethanol series and embedded in Spurr resin (Agar Scientific, Stanstead, UK). Silver/gold sections were cut on a Reichert Ultracut E ultramicrotome (Leica Microsystems, UK), stained with Reynolds lead stain and viewed on a Hitachi H7000 (Hitachi High Technology, Japan) fitted with a SIS Megaview III plate camera (EMSIS, Germany).

The anterior pole of enucleated eyes were removed and posterior eye cups immersed in a primary fixative comprising 3% glutaraldehyde in 0.1 M cacodylate buffer and 2 mM CaCl₂ at pH 7.2. The specimens were then rinsed in 0.1 M cacodylate buffer, 0.23 M sucrose and 2 mM CaCl₂ at pH 7.2, post-fixed in 2% osmium tetroxide in 0.1 M cacodylate buffer for 2 hours, rinsed in buffer, and the block stained with 2% aqueous uranyl acetate. The specimens were dehydrated and embedded in Spurr resin (Agar Scientific, UK). 0.5 μm resin semi-thin sections were cut on a Reichert Ultra cut E ultramicrotome (Leica Microsystems, IL, USA), stained with 1% Toluidine blue and 1% Borax, and viewed under a light microscope. Gold ultrathin sections were cut on the ultramicrotome and stained with Reynolds lead, and viewed on a Hitachi H7000 (Hitachi High Technology, Japan) transmission electron microscope fitted with a SIS Megaview III plate camera (EMSIS, Germany).