ROS formation was determined in 10 µm cryosections of eye globes by dihydroethidium (DHE, 1 µM)-derived fluorescence. In the corneal and conjunctival epithelium, the fluorescence (518 nm/605 nm excitation/emission) was measured as previously described [52]. Moreover, immunostainings for malondialdehyde (MDA), a highly reactive three carbon dialdehyde, which is a marker for oxidative stress, have been performed. Briefly, frozen sections of 10 μm thickness were cut and fixed in 4% paraformaldehyde solution for 20 min. Next, slides were rinsed with PBS and incubated at room temperature with blocking solution containing 1% bovine serum albumin for 30 min. Next, the primary antibody directed against MDA (ALX-210-879, rabbit, polyclonal, Enzo Life Sciences, Inc., Farmingdale, NY, U.S.) was diluted (1:500) in blocking solution and incubated for 2 h at room temperature. Thereafter, each slide was washed in PBS three times for 5 min and incubated for 1 h at room temperature with a Rhodamine Red-X-coupled secondary antibody (111-295-003, goat anti-rabbit, polyclonal, dilution: 1:200, Dianova GmbH, Hamburg, Germany). Negative control sections were incubated with a blocking medium and the secondary antibody. Finally, slides were washed in PBS (3 × 5 min) and were mounted by using VECTASHIELD® Mounting Medium with DAPI (BIOZOL Diagnostica Vertrieb GmbH, Eching, Germany) and cover-slipped. Subsequently, the fluorescence was measured in the corneal and conjunctival epithelium by using Image J (NIH, http://rsb.info.nih.gov/ij/ (accessed on 11 March 2019).

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