Jem 100b electron microscope

For the electron microscopy examination, pieces of the left ventricles of two hearts of each experimental group of animals were taken and fixed in a 2.5% glutaraldehyde solution in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 2 h. After washing with the buffer, the tissue was fixed for 2 h with a 1% solution of osmium acid in PBS and dehydrated by increasing concentrations of alcohols. The resulting samples were encapsulated in Epon 812 resin. Ultrathin sections (70–75 nm) were prepared on a Leica EM UC6 microtome (Leica Microsystems, Wetzlar, Germany) and stained with uranyl acetate and lead citrate. The preparations were viewed and photographed using a JEM-100B electron microscope (JEOL, Tokyo, Japan). Ultrastructural analysis was performed using negative images digitized with an Epson V700 scanner (Seiko Epson Corporation, Nagano, Japan).

For SEM, fixed metamorphic stages of P. harmeri that had been dehydrated in ethanol followed by an acetone series were critical point dried and then sputter coated with platinum-palladium alloy. Specimens were examined with a Jeol JSM scanning electron microscope (JEOL Ltd., Tokyo, Japan).
For TEM, metamorphic stages, newly formed juveniles, and 4-day-old juveniles of P. harmeri were fixed at 4 °C in 2.5 % glutaraldehyde in 0.05 M cacodylate buffer containing 21 mg/ml NaCl and then postfixed in 2 % osmium tetroxide in the same buffer containing 23 mg/ml NaCl. Postfixation was followed by en bloc staining for 2 h in a 1 % solution of uranyl acetate in distilled water. Specimens were then dehydrated in ethanol followed by an acetone series and embedded in Spurr resin (Sigma Aldrich). Semithin and thin sections were cut with a Leica UC5 ultratome (Leica Microsystems GmbH, Wetzlar, Germany). Semithin sections were stained with methylene blue, observed with a Zeiss Axioplan2 microscope, and photographed with an AxioCam HRm camera (Carl Zeiss, Oberkoche, Germany). Thin sections were stained with lead citrate and then examined with a JEOL JEM 100B electron microscope (JEOL Ltd., Tokyo, Japan).