Sample preparation for embedding

The specimens were dehydrated in the ascending graded ethanol solution and kept in a pre-filtration solution for 3 h at room temperature and then in the filtration solution at 4 °C for 17 h. The specimens were then embedded in a light curing resin Technovit 9100 NEW (Kulzer & Co., Wehrheim, Germany) polymerization system based on methyl methacrylate, specially developed for embedding mineralized tissues for light microscopy.

The polymerization mixture was produced by mixing the solution A and B in the proportion of 9 parts A and 1 part of solution B directly before use. This was done in a beaker and using a glass rod to stir the mixture. The samples were then positioned in the labeled plastic moulds, completely covered in the polymerization mixture, and placed in cooled desiccators and under a partial vacuum at 4 °C for 10 min. The resulting blocks were placed in a sealed container and left to polymerize between −8 and −20 °C. The samples were allowed to stand at 4–8 °C in the refrigerator for at least 1 h before allowing it to slowly come to room temperature. The polymerization times are dependent on the volumes of polymerization mixture used and of the constancy of the temperature at which polymerization is carried out.

Preparation of histological sections

The acrylic block was mounted into the object holder of the Leica SP 1600 saw microtome (Fig. 2). The height of the object was adjusted until the surface of the object is slightly above the upper edge of the saw blade. The surface of the block was trimmed to get a plane surface prior to producing slices of a defined thickness. During the sawing process, the water flow was adjusted so that the water jet lands on the edge of the saw blade.