Jm Dumollard

Development of immunostaining of cell cycle related proteins in flat mounted corneal endothelium

Purpose Immunostaining of cell cycle proteins is necessary for the study of the proliferative status of corneal endothelial cells (EC). Most studies use cross sections, which offer direct access to intracellular antigens but allow visualization of only few cells, without giving a global view of an intact endothelium. We developed protocols for immunostaining of ECs of flat mounted corneas Methods Studied proteins: 4 proteins with a known expression pattern (intra-membranous ZO-1, cytoplasmic α actin, nuclear hnRNP and H3 histone) and 8 others, already described within EC: 3 cell cycle inhibitors (P27, P21, P16); 3 proliferation markers (PCNA, MCM2, Ki67), cyclins D1, and E. Fresh and organ cultured (OC) human corneas were used. Fixation/permeabilization: paraformaldehyde (PFA), acetone, methanol, ethanol, Hg, Zn, acetic acid, triton, sodium docecyl sulfate, alone or in combination. Antigen retrieval: heating and biochemical or chemical agents. Patterns of expression were also compared between endothelium and epithelium Results There was no universal protocol. Most of the time 0.5% PFA gave the best results, but for p21, and cyclins, specific protocols were necessary. Homogeneous staining was always obtained with a clear subcellular localization (nuclear or cytoplasmic). OC did not globally modify expression within EC whereas several nuclear or cytoplasmic translocations were observed within the epithelium Conclusion The use of protocols specifically designed for the endothelium of whole intact flat mounted corneas would allow a better localization of cell cycle proteins. They will be especially useful during attempts to alter the cell cycle and trigger EC proliferation. Grant: Fondation de l’Avenir 2007 ET7-468