Immunofluorescence Staining of Corneal Samples

Transverse sections (5 µm thick) of ex vivo or organ-cultured corneas were fixed in 1% vol./vol. formalin or methanol for 5–10 min. LEC cultures were fixed in 10% formalin, permeabilised in 0.2–0.5% Triton X-100 (MilliporeSigma) and blocked in 5% vol./vol. normal goat serum [20 (link)]. Primary antibodies are described in ESM Table 3. AlexaFluor 488- or 594-conjugated secondary antibodies were from Thermo Fisher. All antibodies were diluted in PBS. Slides were mounted using ProLong Gold Antifade mounting medium with DAPI (Thermo Fisher). For each marker, the same exposure time was used when photographing stained sections or cells. Negative controls without a primary antibody were routinely included. Changes in marker expression in individual cases of organ-cultured corneas are indicated in ESM Table 4.