2.7.1. Ultrastructural Investigation by Scanning Electron Microscopy

To analyse their superficial ultrastructure, diaphragmatic tissue specimens were fixed with 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.4) for 24 h, washed 4–5 times with phosphate buffer and gradually dehydrated by successive immersion in increasing concentrations of ethanol for 15 min each. After tissues were critical-point dried and coated with an 8 nm gold layer, specimen observation and micrograph acquisition were carried out by using the tungsten thermionic emission SEM system JSM-6490 (Jeol USA, Peabody, MA, USA).