Ab10353

After treatment for 72 h, the mice were sacrificed, and the eyeballs (n = 3) were enucleated and subsequently fixed in 4% immunohistochemical fixation solution for 2 h. After rinsing with phosphate buffer solution (PBS) three times, all of the corneas were clipped along the margin of the keratosclera and then incubated with anti-β3-tubulin (1:500, ab52623, Abcam, Cambridge, United Kingdom), anti-SP (1:400, ab10353, Abcam) and anti-CGRP (1:400, 14959s, Cell Signaling Technology, United Kingdom) for 48 h at room temperature. After washing with PBS, the cornea was incubated with Alexa Fluor® 488 goat anti-guinea pig IgG H&L (1:500, ab10353, Abcam) and Alexa Fluor® 594 goat anti-rabbit IgG H&L (1:400, ZF-0516, ZSGB-BIO, China) at 4 °C overnight. After counterstaining with 4,6-diamidino-2-phenylindole (DAPI, ZSGB-BIO, China) to stain the nuclei, all of the images were captured by using a fluorescence microscope (Nikon, Tokyo, Japan). To compare the changes in corneal innervation, Image J and Neuron J software were used to calculate the nerve length in the positive area for β3-tubulin staining per sample. In the experiment, one representative image in the center of the cornea and four replicates captured in the peripheral area of cornea from three independent experiments per group were used for analysis.

This assay was performed as previously reported26 (link), with slight modifications. One day before the experiment, HEK293 cells were plated at a density of 5 × 10⁴ cells/well in complete medium. The next day, cells were washed with PBS/0.1% BSA, fixed with paraformaldehyde 4% during 15 min and rinsed with PBS/0.1% BSA. Cell membranes were subsequently permeabilized with 0.1% Triton X100 during 5 min. After a washing step with PBS/0.1% BSA, cells were incubated with the primary monoclonal antibody Ab4E3 (ab10333, Abcam, 5 μg/ml, diluted with PBS/0.1% BSA) or with its isotopic control (Mouse Ig2a kappa Monoclonal, ab10353, Abcam, 25 μg/ml) for 90 min in the dark. Cells were washed twice and incubated for 60 min with goat anti-mouse IgG coupled to FITC (ab6785, Abcam, 1 μg/ml) and with DAPI (Hoechst 33258 pentahydrate (bis-benzimide), H3569, Invitrogen). Finally, cells were fixed a second time with paraformaldehyde 4% during 5 min. Fluorescence was analysed in fluorescent mounting medium (Dako) with a digital Evos microscope (AMG, Westburg).

The tissues were taken 72 h after the injury to the cornea. First, we rewarmed the sagittal frozen sections of corneas at room temperature for 5 min and fixed them with 4% immunohistochemical fixative for 20 min after removing them from the 80 °C temperature. Subsequently, 0.1% Triton solution was added for permeation for 20 min, and 5% BSA (Beyotime, China) was added for 1 h. The cells were incubated with anti-IL-1β (1:200, Abcam), anti-Ki67 (1:200, ab16667, Abcam), anti-SP (1:200, ab10353, Abcam) and anti-CGRP (1:400, 14959s, Cell Signaling Technology) antibodies at 4 °C overnight. Finally, all of the sections were incubated with diluted fluorescent secondary antibody for 1 h. Then, all of the images were captured and analyzed.