Fluorescent microscope

TUNEL staining was performed using In Situ Cell Death Detection kit (Roche Biochemicals, Mannheim, Germany) to detect apoptotic cells, according to the manufacturer’s instructions. Eyes enucleated were fixed in 4% PFA for 24 h at 4˚C. The eyes were then immersed in 25% sucrose for at least 48 h at 4˚C, then embedded in optical cutting temperature (OCT) compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan) and snap-frozen in liquid nitrogen. Cryosections of 10 µm thickness cut on glass slides (FRONTIER COAT; Matsunami Glass Ind, Osaka, Japan) were dried at room temperature and then stored at -80˚C until use. After thawing at room temperature, sections were rinsed in PBS and incubated in 0.1% sodium citrate solution containing 0.1% Triton X-100 for 2 min on ice. They were then placed in the TUNEL reaction mixture at 37˚C for 1 h. After washing with PBS, sections were mounted in Fluoro-keeper antifade reagent non-hardening type with DAPI (Nacalai tesque) and photographed using the fluorescent microscope (Nikon, Tokyo, Japan). The number of TUNEL-positive cells in the retinal outer nuclear layer (ONL) was counted within a compartment 400–700 µm from the optic disc.

Excised ears were fixed using 4% paraformaldehyde (Sigma-Aldrich), treated with xylol (Lach:ner), dehydrated in ethanol, and embedded in paraffin using a Leica EG1150H embedding station. Sections were prepared (8 µm) and stained with hematoxylin and eosin (Leica). Representative images were captured on a Nikon fluorescent microscope with a 20x objective.

The in vitro biocompatibility of hydrogel samples was assessed using L929 mouse fibroblasts. Initially, hydrogel samples with a thickness of 1 mm were prepared in a 24-well culture plate and then freeze-dried and sterilized for the live/dead assay. A suspension of L929 fibroblasts was seeded onto the surface of the scaffolds at a density of 10⁴ cells per well in 100 μl of the culture medium and then incubated for 24 h at 37 °C. Subsequently, the culture media were removed, and the samples were washed with PBS solution. A working solution was prepared by adding 8 μl of fluorescein diacetate (FDA) (5 mg/ml) and 50 μl of propidium iodide (PI) (2 mg/ml) into every 5 ml of PBS to stain viable and dead cells, respectively. The working solution (200 μl) was added to each well, followed by 15 min of incubation, and then washed with PBS again. Finally, live and dead cells, visualized as green and red, respectively, on the scaffold samples were detected using a fluorescent microscope (Nikon, Japan). The experiment was conducted in triplicate.