Materials and Methods
Ten adult Egyptian agama lizards, Trapelus mutabilis , were collected from North Sinai, Egypt, during April and May. Live samples were brought to the Comparative Anatomy of Vertebrates Laboratory and dissected according to the guidelines of the research ethics committee at Aswan University (Approval number: ASWU/05/SC/ZO/23-03/01)\RL.
For anatomical study , the sample heads were fixed in 10% formalin for two weeks and then stored in 2% phenoxyethanol for long-term preservation. Images were acquired using a ToupCam XCAM full HD camera connected to an Olympus microscope (model SZ61). Anatomical terminology of the skull and orbital tissue was determined according to the Nomina Anatomica Veterinaria (2017).
For scanning electron microscopy (SEM) experiments , small pieces of eyes were cut and fixed directly in 5% glutaraldehyde in a cacodylate buffer for 48 hours at 4°C. Samples were washed in three changes of 0.1% cacodylate buffer, post-fixed in a cacodylate-buffered solution of 1% osmium tetroxide for 2 hours at 37°C, and then washed three times in the same buffer. The specimens were dehydrated through an ascending series of ethyl alcohols and infiltrated with amyl acetate for two days. Drying was accomplished by critical point drying using liquid carbon dioxide. Specimens were mounted, sputter-coated with gold, and then examined on a JEOL scanning electron microscope (JSM-5400IV) at 15 kV\RL.
For light microscopy experiments , the lower jaws were removed from the samples, and heads were cut longitudinally into two halves. Specimens were preserved in 10% neutral formalin for three days, decalcified in EDTA for two weeks, dehydrated in a series of ethyl alcohols, cleared in methyl benzoate for three days, embedded in paraffin wax, and serially sectioned at 7 µm. Sections were stained with hematoxylin, eosin, and Masson’s trichromic stains (Drury and Wallington, 1980). Images were acquired using an Olympus camera (model DP74) connected to an Olympus microscope (model BX43).
For transmission electron microscopy (TEM) investigation , small pieces of eyes (1 mm each) were cut and fixed in a cacodylate-buffered solution of 5% glutaraldehyde for 2 hours. Specimens were washed several times in the same buffer for 1 hour at pH = 7.2. Subsequently, the specimens were post-fixed in a cacodylate-buffered 1% osmium tetroxide solution for 2 hours at 4°C. Specimens were washed several times, as in the second step, followed by dehydration in a graded series of alcohol. The specimens were embedded in epoxy resin, treated for semi-thin sectioning at 1 µm thickness, and stained with toluidine blue for light microscopic examination.