Tissue tech o c t compound

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).

Human eyelid tissue, 1–2 days after death was obtained with permission from the University of California Irvine, Willed Body Program and consent of de-identified donors who provided tissue for scientific research. Eyelid tissue was then cut into 2 mm × 5 mm pieces containing the eyelid margin and tarsal plate and remaining, unused tissue was frozen and returned to the Willed Body Program for final disposition. Research tissue was then placed in Tissue Tech O.C.T. Compound (Sakura Finetek USA, Inc, Torrance, CA), snap frozen in liquid nitrogen and stored at −80 °C until cryo-sectioned for immunocytochemistry. Meibomian glands were also dissected from the tarsal plate and RNA and protein extracted for qPCR and western blotting, respectively. For hMGEC, cells were grown in Keratinocyte Serum Free Media (Invitrogen-Gibco, Grand Island, NY) as previously described [9 (link)]. At 80% confluency, cells were passaged onto collagen coated, 60 mm culture dishes and media changed to DMEM-F12 (Gibco, Grand Island, NY) containing EGF (10 ng/ml) and supplemented with either 10% fetal bovine serum (Sigma, St. Louis, MO) or rosiglitazone (30 μM, Sigma). Cells were then cultured for 7 days, and then RNA and protein collected for qPCR and western blotting as discussed below.