IPO13 expression on corneal tissue level compared with other proposal markers. (A): Immunohistochemical or immunofluorescent staining for IPO13, P63, ABCG2, K12, and Cx43 in human corneal and limbal tissues. Limbus shows brighter staining on certain basal cells and no staining on cornea for IPO13, P63, and ABCG2. IPO13 was intensively expressed in nucleus and weakly expressed in cytoplasm of certain basal cells of limbal epithelia. K12 and Cx43 had the similar expression pattern of limbal basal cells showing no staining but the other epithelial cell layers showing bright staining. Brown indicates positive expression by immunohistochemical staining. Red fluorescence indicates positive expression and blue fluorescence indicates nuclear staining with DAPI by immunofluorescent staining. Original magnification: ×200. (B): Double immunofluorescent staining for IPO13 (green) with p63 (red), ABCG2 (red), K12 (red), or Cx43 (red) in the human limbal epithelial cyto-centrifuge slides with DAPI nuclear counter-staining (blue). Coexpression for IPO13 with p63 or ABCG2 is shown by white arrows (B1 and B2). Opposite expression patterns were observed between IPO13 and K12, with Cx43 shown by green arrows (B3 and B4). Original magnification: ×300. (C): Limbal and corneal epithelial explant primary culture at different stages shows that the cell size of cultured limbal epithelia was much smaller than that of cultured corneal epithelial. The density of cultured limbal epithelial cells was increased as the culture time increased and that of cultured corneal epithelial cells decreased; some epithelial cells were detached and some fibroblasts emerged at D14. Original magnification: ×100. (D): Semiquantiative reverse transcription polymerase chain reaction profiles show mRNA expression in limbal and corneal epithelial explant primary culture for IPO13 (478 bp), P63 (440 bp), ABCG2 (379 bp), K12 (150 bp), Cx43 (154 bp), with β-actin (310 bp) as internal control. A 100-bp DNA ladder is shown in the left lane. Original magnification: ×200. Abbreviations: M, DNA markers; L1 and L2, limbal epithelial explant primary cultures; C1 and C2, corneal epithelial explant primary cultures.