Goat anti mouse cd31

The mice were sacrificed on days 7 and 14, and the right eyes were harvested and fixed for 30 minutes at 4 °C in 4% paraformaldehyde. The corneas were separated and blocked with 0.5% Triton X-100 in PBS for 20 minutes at −80 °C and then for 10 minutes at room temperature. Corneas were incubated overnight at 4 °C with goat anti-mouse CD31 (1:20; catalog no. AF3628; R&D Systems, Minneapolis, MN, USA), 2% bovine serum albumin (BSA), 5% normal donkey serum (NDS), and 2% Triton X-100 in PBS. After rinsing in PBS, the corneas were incubated with Alexa Fluor 555-conjugated donkey anti-goat IgG antibody (1:500; catalog no. A-21432; Thermo Fisher Scientific, Waltham, MA, USA) for 2 hours at room temperature. Under the microscope, the corneas were flat mounted on glass slides using mounting medium and coverslips after 3 washes in PBS. In the vehicle control group and the melatonin-treated group, 6 cornea flat mounts were detected at each time point, and in the normal group, a total of 6 corneas were examined. All the flat mounts were captured with a confocal microscope (Carl Zeiss LSM 880, Germany). The degree of CNV was expressed as a percentage of the corneal area by using the confocal microscope built-in software (ZEN 2.3 blue edition, Carl Zeiss Microscopy GmbH, Germany).

Paraffin sections (30 μm) were cut on a HM355S microtome (Thermo Fisher Scientific) and allowed to adhere to Superfrost Plus slides (Thermo Fisher Scientific). Sections were permeabilized and blocked in PBS containing 0.3% Triton X-100 (Sigma-Aldrich) and 10% FBS followed by staining in the same blocking buffer. The following primary antibodies were used for staining: goat anti-mouse CD31 (1:100, R&D Systems); rabbit anti-mouse LYVE1 (1:200, Novus Biologicals); rabbit anti-cKit (CD117) (1:100, Abcam). The following secondary antibodies were used for staining: Alexa Fluor 647 donkey anti-rabbit IgG (H+L), Alexa Fluor 568 donkey anti-goat IgG (H+L) (Thermo Fisher Scientific). Rabbit anti-mouse Glutamine Synthetase (AbCam) was directly conjugated with Zenon Alexa Fluor 488 Rabbit-IgG.
Stained slides were mounted with Fluorescence Mounting Medium (Agilent Dako) and images were acquired on an inverted Leica microscope (TCS STED CW SP8, Leica Microsystems) with a motorized stage for tiled imaging. To minimize fluorophore spectral spillover, we used the Leica sequential laser excitation and detection modality. The bleed-through among sequential fluorophore emission was removed applying simple compensation correction algorithms to the acquired images. Lif files were imported into Imaris (Bitplane) for background adjustment and exported as tiff images.