Sz40 stereomicroscope

The anterior portion of the head was removed from two specimens (181 and 235 mm TL) and fixed in Bouin's fixative [37 ] for 48 h. Samples were then rinsed, stored in 70% ethanol, and decalcified for 48 h in Gooding & Stewart's decalcifying fluid [38 ]. Each sample was divided along the midline into left and right sections and dehydrated through a graded ethanol series (70% to absolute), cleared in xylene and paraffin-embedded. Sagittal sections (5 µm) were obtained using a MicroTec CUT4060 rotary microtome, mounted on glass slides, and stained. We used the Alcian blue-PAS (AB-PAS) stain [39 (link)] to test for the presence of reactive mucopolysaccharides, which would indicate mucus secretion in the RP. This stain has proved to be a reliable technique for detecting glycoproteins in a broad range of animals [39 (link)], including fishes [40 (link)]. We also used the Ayoub–Shklar staining technique to evaluate the occurrence of keratinization in the RP [41 (link)]. Photomicrographs were taken using an Olympus SZ40 stereo microscope equipped with an SZ-CTV adapter and an Olympus DP21 digital camera. The thickness of the keratinized layer was measured at 10 haphazardly selected points along the RP to estimate its average thickness (using Adobe Illustrator CC 2017).

GUS staining of transgenic plants was performed as described before (Calonje et al., 2008 (link)). Briefly, the tissue was incubated in 2-mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid in 50-mM phosphate buffer, pH 7.0, containing 0.5-mM K₃Fe(CN)₆ and 0.5-mM K₄Fe(CN)₆ for 2 h at 37°C. The tissue was then rinsed with 50-mM phosphate buffer and fixed in ethanol (95%):acetic acid (9:1, v/v) for 2 to 4 h at room temperature. Images were captured under an Olympus SZ40 Stereo Microscope.