Rabbit anti perilipin

Freshly dissected femurs were fixed in 4% paraformaldehyde overnight followed by 3-day decalcification in 10% EDTA. O.C.T. (SciGen)-embedded bones were sectioned using the CryoJane Tape-Transfer system (Leica Biosystems). Sections were blocked in PBS with 5% donkey serum for 1 h and then stained overnight with rabbit-anti-perilipin (Sigma, 1:800, P1873), goat-anti-ENDOGLIN (R&D, 1:500, AF1320), rabbit-anti-TRAP (Abcam, 1:200, ab185716) or rabbit-anti-AGGRECAN (Millipore, 1:400, AB1031). Donkey anti-rabbit Alexa Fluor 647 or donkey anti-goat Alexa Fluor 647 were used as secondary antibody (both from Invitrogen, 1:500). Non-immune immunoglobulins of the same isotype as the primary antibodies were used as negative controls. Slides were mounted with anti-fade solution (Invitrogen). Images were acquired with a Zeiss LSM880 or a Leica SP8 confocal microscope.

Antibodies used include chicken anti-GFP (Aves, GFP-1020), rabbit anti-Perilipin (Sigma, P1998), and rat anti-CD31 (BD Bioscience, 553370). Mice were transcardially perfused with PBS followed by 4% formaldehyde in PBS. Tissues were excised and fixed overnight, cryoprotected in 30% sucrose in PBS, then frozen in OCT (Sakura), and stored at −80°C. Cryosections (10 μm) were prepared using a Leica cryostat and mounted on SuperfrostPlus sides (Fisher). After drying, sections were blocked with 5% bovine serum albumin, followed by labeling with primary antibody for 1 hr at room temperature. After five washes with PBS/0.1% Triton X-100, sections were incubated with appropriate secondary antibodies (goat antichicken Alexa 488, A11039; goat antirat Alexa 647, A21247; goat antirabbit 647, A21244; all from Invitrogen) for 30 min at room temperature. Following additional washes, coverslips were mounted with DAPI antifade reagent (Vector Labs) and sealed with clear nail polish. Images were obtained using inverted Leica epifluorescence microscope, pseudocolored using ImageJ, and merged in Photoshop CS4 (Adobe Creative Suites).