Col1a1

For IHC, frozen sections were washed with 1X PBS three times followed by incubating with 0.3% H₂O₂/0.3% horse serum in 1X PBS for 5 minutes at RT. Then, slides were washed in TNT buffer (0.1M Tris pH 7.5, 0.15M NaCl, 0.05% Tween-20) 3 times. Slides were then blocked one hour with TNB buffer (0.1M Tris pH 7.5, 0.15M NaCl, 0.5% blocking reagent) at RT, followed by incubation with primary antibody (COL1A1, Rockland, 1:1000) diluted in TNB buffer overnight at 4°C. Slides were then washed with TNT buffer 3 times, incubated with Horseradish peroxidase (HRP) conjugated secondary antibodies (Life Technologies) for 2 hours at RT, signals were developed by using the DAB staining kit (Vector Laboratories, Cat.# SK-4100). For IF staining, frozen sections were washed in PBS, and then blocked by 5% donkey serum in PBST for one hour at RT, followed by incubation with primary antibodies (α-SMA, 1:3000; COL1A1, Rockland, 1:1000; KLF4, Cell Signaling Technology, 1:50; FBXO32, 1:100, ECM Bioscience; PDGFRB, 1:50, Cell Signaling Technology) overnight at 4°C. Alexa Fluor 488 or 568-conjugated secondary antibodies (Donkey anti-Mouse IgG, or Donkey anti-Rabbit IgG or Donkey anti-Goat IgG; Life Technologies, 1:250 dilution) were used for the visualization of signals. Nikon DS Ri1 and SPOT software were used for the image acquisition and analysis.

Tissues were fixed in 4% paraformaldehyde for 48 h, incubated in 15% DEPC-EDTA (pH 7.8) and ultrasonically decalcified. The specimens were embedded in paraffin or OCT and cut into 7 μm sections.
Immunofluorescence assay: Sections were blocked in PBS with 10% horse serum and 0.1% Triton for 1 h at room temperature. Then, the cells were stained overnight with rabbit anti-perilipin A/B (Sigma, P1873, 1:1 000, USA) and OPN (1:1 000; R&D, AF808). Donkey-anti-rabbit Alexa Fluor 488 (1:1 000; Molecular Probes, A21206) and donkey-anti-goat Cy3 (1:1 000; Jackson ImmunoResearch, 705–165–147) were used as secondary antibodies. DAPI (Sigma, D8417) was used for counterstaining. Slides were mounted with anti-fluorescence mounting medium (Dako, S3023), and images were acquired with an Olympus FV3000 and SP8 confocal microscope.
Immunohistochemical staining and Col1a1 (1:100; Rockland, 600–400–103) staining were performed as described by Dako.
Tissue sections were used for TRAP, BODIPY, and Oil Red O staining according to the standard protocol.