Ab66440

The eyes were harvested on the 7th day after alkali injury and embedded in optimal cutting temperature (OCT) compound after the fixation with 4% paraformaldehyde overnight at 4°C. We blocked 5 μm OCT frozen sections with 3% BSA for 1 h at room temperature and incubated the sections overnight at 4°C with a rat anti-F4/80 antibody (Abcam, ab66440, 1:200), rat anti-CD11b antibody (Abcam, ab8878, 1:200) or rabbit anti-CD31 antibody (Abcam, ab28364, 1:100). Detection of bound anti-F4/80, anti-CD11b or anti-CD31 antibodies was performed using anti-rat IgG Alexa Fluor 488 (Cell Signaling Technology, Germany, 1:1000), anti-rat IgG Alexa Fluor 555 (Cell Signaling Technology, Germany, 1:1000) or anti-rabbit IgG Alexa Fluor 555 (Cell Signaling Technology, Germany, 1:1000). After counterstaining with DAPI (Abcam, ab228549), the sections were mounted with antifade mounting medium (Beyotime, Shanghai, China). The sections were viewed and photographed with fluorescence microscopy (DMI3000 B; Leica, Wetzlar, Germany).

For the corneal inflammatory cell recruitment test, mouse eyes were embedded in a compound at the optimal cutting temperature, sequentially sectioned (8 µm) and stored at −80°C. Subsequently, the frozen sections were blocked with 3% bovine serum albumin at 37°C for 1 h. Then at 4°C, the frozen sections were inducible rat anti-F4/80 (Abcam, ab66440, 1:100) and rat anti-CD11b (Abcam, ab8878, 1:100) overnight. Sections were then washed with phosphate buffered saline and incubated with anti-rat IgG Alexa Fluor 555 (cell signaling technology, 1:1,000) and anti-rat IgG Alexa Fluor 488 (Cell signaling Technology, Germany, 1:1,000) for 1 h at room temperature, followed by DAPI (Abcam, Ab228549) solution staining. Photographs were taken with a fluorescence microscope (DM 4000B). The number of positive cells was quantified using Adobe Photoshop CC (Adobe Systems, Inc., San Jose, CA, United States) with the method described by et al. (Dai et al., 2018 (link)). All histological assessments were performed as blinded studies by the same two observers.