Nlo780

At 14d, 21d and 28d post-injury, mice were deeply anesthetized and fixed on a designated plate, with the ocular surface faced up. According to a previously described method [7 (link)], a modified plastic bowl containing sterile PBS was fixed on the ocular surface sealed by erythromycin eye ointment to ensure the use of water immersion objective. SHG imaging was performed using an inverted two-photon excitation fluorescence microscope (NLO780, Zeiss). The laser was tuned to 780 nm and a 20× water immersion objective (numerical aperture =1.0) was used to focus the excitation beam and to collect backward signals. The cornea was scanned layer by layer using Z-stack (Z = 5 μm), and the obtained images were three-dimensionally reconstructed using Imaris software (× 64, version 7.4.2, Bitplane, Zurich, Switzerland) to calculate the average signal intensity of the image. The stronger the image signal intensity, the more regular the corneal matrix collagen fibers are arranged, and conversely, the weaker the image signal, the corneal matrix collagen fiber structure is disordered or degraded.

Immunostaining of cells or ECM was performed as described21 (link). Fluorescence was observed through × 40/1.3 or × 63/1.4 oil objectives on a Zeiss inverted epi-microscope (Axiovert 200 M) equipped with an ANDOR NEO sCMOS camera. Second-harmonic generation and confocal imaging were performed on a multi-photon microscope (Zeiss NLO780) confocal system with a × 63/1.4 objective.

The cells were fixed in 4% paraformaldehyde for 10 min, followed by washing in PBS, permeabilized with 0.1% Triton-100 for 10 min and blocked in 5% FBS for 30 min. Then incubated cells with anti-flag (1: 200; ab205606, Abcam, USA) and anti-COXIV (1: 100; 11967S, Cell Signaling Technology, USA) antibodies overnight at 4 °C. After being washed with PBS three times, secondary anti-mouse Alexaflor 594 (1:250; abs20017, Absin, China) and anti-rabbit IgG-FITC antibodies (1:250; abs20004, Absin, China) were then applied for 2 h at room temperature. Nuclei were stained with DAPI. Zeiss confocal microscope (Zeiss NLO780; Zeiss, Germany) was used to obtain fluorescence microscopy images.