The investigation of the distribution of nerves, blood vessels and immune cells on the fresh human corneal surface using optimized protocols for immunostaining of flat mounted whole cornea

• Published in: Acta ophthalmologica (Oxford, England) Vol. 95; no. S259
• Main Authors: He, Z., Guindolet, D., Forest, F., Cognasse, F., Acquart, S., Perrache, C., Gabison, E., Bergandi, F., Gain, P., Thuret, G.
• Format: Journal Article
• Language: English
• Published: Malden Wiley Subscription Services, Inc 01.09.2017
• Subjects: Actin; Basal cells; Blood vessels; Capillaries; CD4 antigen; CD45 antigen; CD8 antigen; Cell culture; Chromatin; Conjunctiva; Cornea; Cytokeratin; Cytoplasm; E-cadherin; Epithelium; FDA approval; Fibers; Fixatives; Histone H3; Localization; Methanol; Nerves; Neurofilaments; Pax6 protein; Preservation; Proteins; Tubulin; Zonula occludens-1 protein
• ISSN: 1755-375X; 1755-3768
• DOI: 10.1111/j.1755-3768.2017.0T016

Purpose To present a reliable protocol for the immunostaining of flat mounted corneas and provide a map of the distribution of nerves, blood vessels and immune cells on fresh human corneal epithelium and conjunctiva. Methods Organ‐cultured human corneas were used for our technique development. 15 fixatives and 5 antigen retrieval (AR) methods were assessed using 4 ubiquitous proteins with different, well‐characterized subcellular localization: ZO‐1 (membrane), actin (cytoplasm), hnRNP L (nucleosol) and histone H3 (chromatin). The distribution of nerves (Neurofilaments and tubulin β3), blood vessels (CD31 and α‐SMA) and immune cells (CD45, CD4, CD8, CD68) in fresh human corneas (without any preservation in culture medium) was then investigated using these optimized protocols. Results 0.5% paraformaldehyde (PFA) and pure methanol were selected as the most efficient fixatives. Using our optimised protocol, we revealed not only superficial cells (cytokeratin 3, E‐cadherin), but also basal cells (ΔNp63, PAX6). Nerve fibers were mainly stained by tubulin β3 on epithelium and by Neurofilament on conjunctiva. CD31 stained only the capillaries situated at the limbus, whereas α‐SMA stained all types of vessels on the corneal surface. Numerous immune cells were detected on conjunctival and capillary loops below the limbus. A few immune cells were observed on central epithelium. Conclusions Tissue fixation with 0.5% PFA or pure methanol, without any subsequent AR techniques, were found to produce the best results. Compared to IHC on cross sections, immunostaining of flat mounted cornea allows a precise localization of targeted cells through the full thickness of the corneal epithelium and conjunctiva. The distribution of nerves, blood vessels and immune cells on the corneal surface has therefore been mapped.