Rabbit perilipin antibody

Isolated long bones were fixed in 4% paraformaldehyde, decalcified using 0.5 M EDTA in phospate buffered saline (PBS) then vibratome (100‐200 μm) or frozen (20‐25 μm) sectioned. Immunofluorescence was performed using chicken or rabbit GFP antibody (Abcam, 1:1000), rabbit osterix antibody (Abcam, 1:600), rat CD31 antibody (Pharmingen, 1:200), rat CD45 antibody (Pharmingen, 1:100), rabbit perilipin antibody (Sigma, 5 μg/mL), rabbit mTert antibody (Millipore, 1:150) or rabbit osteocalcin antibody (Abcam, 10 μg/mL) and Alexa Fluor 488, 594, 633, or 647 secondary antibodies (Molecular Probes, 1:400). LipidTOX Deep Red (ThermoFisher, 1:200) was used for lipid staining. Nuclei were stained using DAPI. Sections were imaged using either a 90i Eclipse (Nikon) or LSM 700 laser scanning confocal (Zeiss) microscope.

Long bones were collected and the BM was flushed by centrifugation to obtain marrow stromal cells.³⁵ Following red blood cell (RBC) lysis, 10⁵ to 10⁶ cells were plated in MesenCult MSC Basal Medium supplemented with MesenCult Mesenchymal Stem Cell Stimulatory Supplement (STEMCELL Technologies), glutamine and antibiotics. Adherent cells were analyzed after 24 hours and colonies were visualized after 2 weeks. To promote differentiation, cells were cultured to confluence and then the media was supplemented with either 50 μg/mL ascorbic acid (Sigma), 7 mM β‐glycerophosphate (Sigma), 10⁻⁸ dexamethasone (Sigma) for osteoblastogenesis, 20 ng/mL TGF‐β3 (PeproTech), 200 mM ascorbic acid for chondrogenesis or Adipogenic Stimulatory Supplement (Stem Cell Technologies) for adipogenesis. Differentiation media was changed twice a week and cells were cultured up to 4 weeks. Immunofluorescence was performed using rabbit perilipin antibody (Sigma, 5 μg/mL), goat collagen II antibody (Santa Cruz, 1:250) or rabbit osterix antibody (Abcam, 1:600) and Alexa Fluor 350, 633 or 647 secondary antibodies (Molecular Probes, 1:400) and imaged using an inverted Eclipse Ti (Nikon) or LSM 700 laser scanning confocal (Zeiss) microscope. Nuclei were stained using 4′,6‐diamidino‐2‐phenylindole (DAPI) (Sigma, 1:1000).