Sc 365578

Hematoxylin and eosin staining was performed by fixing enucleated eyeballs in 4% paraformaldehyde as described earlier (Starr et al., 2019 (link); Saltykova et al., 2021 (link)). Cryopreserved eyes were sectioned with 12-μm thickness using the Leica CM1510S cryostat (Leica, Buffalo Grove, IL). Corneal thickness was counted in 20× magnification with a 200-µm step distance between points. The vehicle-treated left eyes and NM-exposed right eyes were used for corneal morphometry.
The IHC analysis was conducted on the corneal sections using an anti-VEGF antibody (dilution 1:100; Santa Cruz Biotechnology, sc-365578). Donkey anti-Mouse IgG and Alexa Fluor 555 were employed as secondary antibodies (Invitrogen, PIA32773). Imaging analysis was carried out using the BZ-X800 fluorescence microscope (Keyence, Itasca, IL). To quantify the intensity of VEGF staining, two randomly selected fields at the center of the corneas were used. ImageJ software was employed to calculate the ratios of integrated density of VEGF signal over the area of interest (n = 6). A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was described in our previous study (Zhylkibayev et al., 2023 (link)). TUNEL-positive nuclei were counted manually by masked investigators (n = 7).

Proteins were extracted from cells cultured in RIPA lysis buffer (RIPA, China) as described in the RT-PCR protocol. One SDS-PAGE gel was loaded with 50μg of protein per well. After the proteins were transferred on nitrocellulose membranes, antibodies specific for Dll4 (1:200, SC365429, Santa Cruz, CA), Notch1 (1:200, SC376403, Santa Cruz, CA), Notch4 (1:200, SC393893, Santa Cruz, CA) and the VEGF (1:200, SC365578, Santa Cruz, CA) were used for incubation. Band intensity was analyzed with a gel imaging analysis system (Image Lab, Bio-Rad, USA).