Immunohistochemical Analysis of Corneal Tissue

Immunohistochemistry was carried out as previously described [25 (link)]. Briefly, corneoscleral tissue samples and organ-cultured corneoscleral tissue samples were frozen in liquid nitrogen after being embedded in an optimal cutting temperature (OCT) compound. Cryosections of 6–8 μm thickness were cut from the superior or inferior quadrants, fixed in 4% paraformaldehyde for 15 min, and blocked with 10% normal goat/donkey serum (NGS/NDS) in 0.3% Triton X−100 in PBS. Then, the samples were incubated in primary antibodies (Supplementary Table S1) diluted in 2% NGS/NDS, 0.1% Tween20, in PBS overnight at 4 °C or 2 h at room temperature. Alexa-488-,568-,558-conjugated secondary antibodies (Life Technologies, Carlsbad, CA, USA) were used to detect antibody binding, and the sections were mounted in Vectashield antifade mounting media with DAPI (Vector, Burlingame, CA, USA). A laser scanning confocal microscope (TCS SP−8, Leica, Wetzlar, Germany) was used to examine immunolabeled samples.