H 300 transmission electron microscope

Images were acquired with a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany; objectives 10×, 20× and 40× Leica). The ultra-thin sections were examined with a Hitachi H-300 Transmission Electron Microscope. The pictures (enlargement 40×) obtained by immunohistochemistry with anti- HABP were analyzed by conversion to 8-bit, inverted. The diameter of the cytoplasmic and released vesicles (120 vesicles for time) was calculated by ImageJ software (freely available at http://rsb.info.nih.gov/ij/, 5 July 2022). The modal grey values of each vesicle, and of the cytoplasm of its origin cell, were calculated. In the cytoplasm, we calculated the model grey value of at least three different areas of cytoplasm per each cell and calculated the mean. The ratio of each vesicle to the mean of the cytoplasm was then calculated. Finally, the mean ratio with standard deviation was obtained.

Two specimens from each of the human male groups (young and elderly) were randomly selected and fixed in 2.5% glutaraldehyde (Serva Electrophoresis, Heidelberg, Germany) in 0.1 M phosphate-buffered, post-fixed in 1% osmium tetroxide (OsO4) (Agar Scientific Elektron Technology, Stansted, UK) in 0.1 M phosphate buffer, dehydrated in a graded ethanol series, and embedded in Epoxy Embedding Medium Kit (45349, Sigma-Aldrich, St. Gallen, Switzerland). Ultrathin (60 nm) and semi-thin (0.5 µm) sections were cut with a RMC PowerTome ultramicrotome (Boeckeler Instruments, Tucson, AZ, USA) and stained with 1% toluidine blue at 80 °C. The ultrathin sections were collected on 300-mesh copper grids and counterstained with 1% uranyl acetate and then with Sato’s lead solution. The specimens were examined using a Hitachi H-300 Transmission Electron Microscope.