Goat anti rat alexa fluor 555

Mice were sacrificed on day 7 after corneal AB for F4/80 immunostaining and on day 14 for CD31 immunostaining. The eyes were removed, fixed with 4% paraformaldehyde (PFA) overnight at 4 °C, and embedded in OCT. Sagittal sections (8 µm) were cut and blocked with 5% bovine serum albumin (BSA) in a Tris-buffered saline (TBS) buffer for 1 h at room temperature. Then, the sections were incubated with rat anti-F4/80 (Cat. No. Ab6640, 1:100, Abcam, Cambridge, UK) or rabbit anti-CD31 (Cat. No. Ab28364,1:50, Abcam) primary antibody overnight at 4 °C and further incubated with goat-anti-rat Alexa Fluor 555 (Cat. No. 4417, 1:1000, Cell Signaling Technology (CST), Danvers, MA, USA) or goat-anti-rabbit Alexa Fluor 555 (Cat. No. 4413, 1:1000, CST) secondary antibody for 1 h at room temperature. After counterstaining with DAPI (Abcam), the sections were examined with a fluorescence microscope (Nikon, Tokyo, Japan). We recognized the CD31⁺DAPI⁺ or F4/80⁺DAPI⁺ double immunostaining cells in the corneal stroma and count them at 20× magnification in five different fields of each immunostaining section manually. For each sample, three planes were selected evenly within the pupillary zone. Subsequently, three sections were selected from each plane. The counting work was all performed by the same two observers not aware of the experimental design and groups of study [25 (link)].