S6d stereomicroscope

The stamen primordia and gynoecium primordia were dissected carefully and photographed using a LEICA S6D stereomicroscope (Leica Microsystems, Wetzlar, Germany). Then, the floral buds, stamen primordia, and gynoecium primordia were fixed in FAA for 24 h for SEM and paraffin sectioning. Some fixed materials were dehydrated in an ethanol series for 20 min per step and dried using CO₂ as the exchange agent by an EMITECH K850 critical point dryer (Emitech, Canterbury, British). Then, the samples were coated with gold by an Edwards E-1010 ion sputter golden coater (Hitachi, Tokyo, Japan) and photographed with an FEI Quanta 200 scanning electron microscope (FEI, Eindhoven, Netherlands).
The remaining fixed materials were dehydrated in an ethanol series, transparentized in a xylene series, infiltrated in paraffin, embedded in paraffin wax, and sectioned at an 8 µm thickness with a LEICA RM2145 rotary microtome (Leica Microsystems, Wetzlar, Germany). Finally, the sections were stained with safranin and fast green and photographed with a Zeiss AXIO Axioscope A1 fluorescence microscope (Carl Zeiss, Jena, Germany).

Upon topical anesthesia, each treated eye was examined with a stereo biomicroscope before application of topical treatment at 30, 54, 78, 100, and 172 h after lesion (also referred to in this study as day 1, 2, 3, 4, and 7, respectively). This was done at each time point to assess corneal inflammation, opacities, and other anterior surface complications (i.e., infection, perforation, etc.). Fluorescein sodium solution (Colircusí Fluoresceína, Alcon Cusí, Barcelona, Spain) was used to evaluate the degree of the corneal epithelial defect. Each animal’s anterior segment was photographed with a Leica S6D stereo microscope (6.3:1 zoom and 15.0× magnification) equipped with a Leica EC3 digital camera (Leica Microsystems, Wetzlar, Germany) with and without fluorescein at each clinical assessment. The defect area was determined by the fluorescein positive remaining area under blue light (1 mm = 240 pixels) using ImageJ 1.45a software (National Institutes of Health, Bethesda, MD, USA). Based on anterior segment visualization of pupil, iris and the presence of corneal vessels with stereo biomicroscopy at each examination time point. The analysis was performed independently by two masked graders.