S 3000n scanning

For electron microscopy, 10⁵ germlings/mL of strains Af293 and CEA10 were incubated with A549 and HSAE cells on glass coverslips for 2.5 h. The coverslips were then rinsed with PBS and fixed overnight with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4°C. After dehydrating and critical-point drying the samples, they were sputter coated with Au-Pd and imaged with a Hitachi S-3000 N scanning electron microscope.

The external morphology of specimens was examined from photographs of living Okenia specimens and from laboratory observations. Internal organs were removed following a dorsal incision and drawn using a Nikon SMZ-1500 dissecting microscope with an attached camera lucida. Special attention was paid to the buccal mass and the reproductive system. Each buccal mass was removed and dissolved in 10% sodium hydroxide to remove surrounding tissue. The labial cuticle and radula were then rinsed in water. These structures and the penis were initially examined under the light microscope, then photographed using the software cellSense, and subsequently dried (apart from the radula) by critical point using hexamethyldisilazane. All these parts were finally mounted and sputter coated for examination under a Hitachi S3000N scanning electron microscope (SEM).

The samples prepared for electron microscopy were fixed overnight at 4 °C with 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.4), and then the samples were dehydrated in a graded ethanol series followed by substitution with a graded isoamyl acetate series. The samples were critical point dried, sputter coated with gold and observed using a HITACHI S-3 000N scanning electron microscope at 10kV. For transmission electron microscopy analysis, samples were fixed overnight at 4 °C with 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.4). After washing with phosphate buffer three times, samples were fixed overnight in 2% (v/v) OsO4 in phosphate buffer. After staining and dehydration in a grade ethanol series, the samples were submerged with LR White resin and polymerized for 2 d. Ultrathin cross-sections were prepared with a Leica EM UC6 ultra-microtome and observed under a Tecnai Spirit (120kV) transmission electron microscope.

For the SEM analysis, the cell-seeded membrane was removed from the cell culture plate and fixed with glutaraldehyde for 3 h at room temperature. All of the samples were washed 3 times with PBS and distilled deionized water, and were treated with increasing concentrations of ethanol from 50% to 99.5% for dehydration. The samples were air dried and sputter coated with gold for observation with a Hitachi S-3000N scanning electron microscope (SEM, Tokyo, Japan).