Rabbit anti pparγ

After rats were perfused with ice-cold PBS (0.1M, pH 7.4) at 24 h post-operation, the ipsilateral cortex were collected and stored in −80 °C freezer until use. Western blot was performed as described previously (Tong et al., 2017 ). After protein samples preparation, equal amounts of protein (50 µg) were separated by SDS-PAGE gel electrophoresis, and then transferred onto nitrocellulose membranes. Membranes were blocked and incubated with the following primary antibodies overnight at 4 °C: rabbit anti-NTN-1 (1:800, Abcam, USA), rabbit anti-UNC5B (1:1000, Abcam, USA), rabbit anti-PPARγ (1:500, Abcam, USA), rabbit anti-NFκB P65 (1:1000, Abcam, USA), mouse anti-IL-6 (1:1000, Abcam, USA), goat anti-TNF-α (1:1000, Abcam, USA), rabbit anti-ICAM-1 (1:1000, Santa Cruz Biotechnology, USA), and rabbit anti-myeloperoxidase (MPO, 1:1000, Santa Cruz Biotechnology, USA). β-actin was used as the internal loading control. Then, membranes were incubated with horseradish-peroxidase conjugated secondary antibodies (Santa Cruz Biotechnology, USA) for 1 h at room temperature, The immunoblots were probed with an ECL Plus chemiluminescence reagent kit (Amersham Biosciences, USA). The relative density of protein was analyzed by ImageJ software (ImageJ 1.5, NIH, USA).

For analysis of meibomian glands, eyelids from 3 young (2-month-old) and 3 old (2-year-old) mice was initially fixed overnight in 2% paraformaldehyde in PBS at 4°C, washed in PBS, embedded in Tissue Tech O.C.T. Compound (Sakura Finetek USA, Inc, Torrance, CA), frozen in liquid nitrogen and stored at −80°C and then sectioned using a Leica CM1850 Cryotome (Leica, Wetzlar, Germany), and mounted onto glass slides. For analysis of cultured meibocytes, cells were fixed in 2% paraformaldehyde in PBS for 2 hours.
For immunostaining, cells and tissue sections were permeablized in PBS containing 0.5% dimethyl sulfoxide and 0.5% Triton X (pH 7.2) for 5 minutes and then washed in PBS. Slides were then incubated in goat serum (1/30) for 30 minutes at 37°C and then incubated with rabbit anti-PPARγ (1:50, Abcam, Cambridge, MA). Slides were then washed with PBS, stained with FITC conjugated goat anti-rabbit IgG (1:200, Invitrogen) for 1 hour at 37°C and then counterstained with DAPI (Invitrogen). For negative controls, primary antibodies were either omitted or replace by nonspecific rabbit sera. The samples were then evaluated and imaged using a Nikon Eclipse E600 epifluorescence microscope (Nikon Inc, Melville, NY).