Ms 145 p0

To evaluate the localization and expression levels of Nrf2, Muc1, and Muc5ac in the ocular surface, fluorescent immunohistochemical analysis was performed as follows. Briefly, 6-mm cryosections from the mouse eyeball were fixed in 4% paraformaldehyde for 20 minutes. After blocking with 1% bovine serum albumin in phosphate-buffered saline containing 2% donkey serum, sections were incubated overnight with primary antibodies. After washing with phosphate-buffered saline, the sections were incubated for 30 minutes with secondary antibodies and were observed using a fluorescence microscope (Carl Zeiss Inc., Oberkochen, Germany). For negative controls, isotype control IgG was applied instead of primary antibody. The specimens were immunostained using the following primary antibodies: rabbit anti-Nrf2 antibody (0.01 mg/mL, ab31163; Abcam, Boston, MA), mouse anti-Muc5ac antibody (2 mg/mL, MS-145-P0; Thermo Fisher Scientific Inc., Cheshire, UK), and rabbit anti-Muc1 (0.2 mg/ mL, ab8878; Abcam). The secondary antibody was fluorescein isothiocyanateeconjugated anti-rabbit IgG antibody (0.0075 mg/mL; Jackson ImmunoResearch Laboratories, West Grove, PA). DAPI (Vector Laboratories) was used for nuclear staining.