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.