Expression of OCTN2 and ATB0, + in human corneal epithelial cells and corneal tissues

The expression of OCTN2 and ATB^0,+ in HCECs was observed by immunofluorescence. HCECs were seeded on a 12-mm coverslip in 24-well plat at 1 × 10⁵ cells/well density. After cells grew to 90% confluence, the cells were fixed with 4% paraformaldehyde for 20 min and blocked with 3% BSA solutions for 30 min. Then, the primary antibody against ATB^0,+ and OCTN2 was added to the cells and cultured for 12 h at 4 °C. Alexa Fluor 594 labeled secondary antibody was added to the cells and incubated for 60 min at 37 °C. The cell nucleus was stained with DAPI. Lastly, the cells were observed by fluorescence microscope (Carl Zeiss, Germany).

The expression of ATB^0,+ and OCTN2 in rabbit corneal tissue was also examined by immunofluorescence analysis. The rabbits were euthanized with air injection. The corneal tissues were excised and rinsed with ice-cold pH 7.4 phosphate-buffered saline. For immunofluorescence analysis, the corneal tissues were fixed with 4% paraformaldehyde and paraffin sections of 10 μm were prepared. The sections were blocked with 3% BSA solutions for 1 h. The samples were stained with rabbit anti-ATB^0,+ antibody and rabbit anti-OCTN2 antibody for 12 h. Lastly, the corneal tissues were observed by fluorescence microscope (Carl Zeiss, Germany).