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Zhiguo HE, Chantal PERRACHE, Fabien FOREST, Damien Guindolet, Eric Gabison, Fabrice COGNASSE, Florian BERGANDI, Sophie ACQUART, Philippe GAIN, Gilles Thuret; Optimized protocols for immunostaining of the epithelium of flat mounted whole human corneas. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2615. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Immunostaining on flat mounted human corneal epithelium presents numerous advantages over classical immunohistochemistry on cross sections. Nevertheless, there are no well-defined and reproducible protocols. Aim: to present a few reliable protocols to maximize the staining quality of through-thickness of the corneal epithelium.
Thirty human corneas stored in organ culture (OC) (CorneaMax, Eurobio), and 12 fresh corneas (body donation to Sciences) were used. The mean donor age was 72±6 years old. Corneas were divided in 6 to 8 pie-shaped pieces to increase the number of samples. The first test consisted in selecting among 15 fixative and 5 antigen retrieval (AR) methods. Four ubiquitous proteins with different, well-characterized subcellular localization were chosen: ZO-1 (membrane), actin (cytoplasm), hnRNPL (nucleosol) and histone H3 (Chromatin). The best protocols were then tested with a large series of antibodies targeting proteins of potential interest: differentiation and stem cell markers (K3/K12, E-cadherin, ΔNp63, PAX6, ABCB5, N-cadherin…), nerves markers (tubulin βIII, NCAM, neurofilament, GFAP, Synaptophysin…), immune cells (CD3, CD4, CD8, CD14, CD20, CD45, CD56, CD68, Ps100…) and growth factor receptors (EGFR, FGFR, IGFR, NGFR, TGF-βRs…).
0.5% paraformaldehyde (PFA) and pure methanol were the most efficient fixatives (especially better than standard 4%PFA). 0.5% SDS was the most efficient AR method. Using optimised protocols, we revealed not only the superficial cells (K3/K12, E-cadherin) but also basal cells (ΔNp63, PAX6) that were not restricted to the limbus, but distributed everywhere in the basal epithelium. Nerve fibers were clearly stained by tubulin βIII, NCAM and neurofilament with different distributions on epithelium and conjunctiva. Numerous leukocytes (CD45+) were detected at basal level of peripheral epithelium and few in the center. They were cytotoxic T cells (CD3+, CD8+), dendritic cells (Ps100+), macrophages (CD68+) and natural killer cells (CD56+). EGFR, IGFR and NGFR were mainly expressed on the membrane of the basal epithelial cells.
These optimized protocols for immunostaining of the epithelium of flat mounted whole human corneas may be a powerful tool for epithelial investigations by providing not only a precise localization of targeted cells in the whole epithelium and limbus, but also a clear subcellular localization of proteins.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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