Dissection and histology of mental glands

Mental glands from four adult turtle specimens (three males and one female) from CRARC were used for histological examination. MGs were dissected from freshly dead turtles and stored in chemical buffers (see below). Turtles died from trauma-related injuries and the necropsy procedure was performed at CRARC by a specialized veterinarian (A Martínez-Silvestre). MG structure was examined using LM and TEM.

For examination in LM, entire MGs were fixed in Bouin’s solution immediately after excision. Dehydration was done in an alcohol gradient followed by clearing in xylene and embedding in paraffin as previously described (Piprek et al., 2012). Sectioning of paraffin blocks was conducted on a ZEISS HYRAX microtome. Paraffin sections (6 μm thick) were stained with Harris’s hematoxylin and eosin to visualize the general structure of the glands. Alcian blue staining was used to detect acidic polysaccharides. Periodic acid-Schiff (PAS) stain was used to detect polysaccharides. Mallory’s trichrome stain was used for the visualization of collagen (Kiernan, 1990).

Small fragments of MGs used for TEM were fixed in Karnovsky’s fixative (Karnowsky, 1965). The material was washed in 0.1 M cacodylate buffer and post-fixed in 1% osmium tetroxide. Dehydration was carried out in a series of graded ethanol solutions. Then the material was embedded in epoxy resin (EPON-812) as previously described (Piprek et al., 2017). Semithin sections (0.5 μm) of the resin were stained with methylene blue and Azure II (1:1) for LM examination. Ultrathin sections (60–70 nm) were contrasted with uranyl acetate and lead citrate for TEM examination. Images were collected with a JEOL-2100HT transmission electron microscope in the Department of Cell Biology and Imaging, Jagiellonian University (Kraków, Poland).

LM and TEM images were processed in CorelDRAW and Corel Photo-Paint to create the layout of the figures. Basic adjustments on image brightness, contrast, intensity and tone were performed if needed.