Ab23914

Different types of conditioned media were prepared and collected, which were stored at -80°C after a centrifugation at 2500 rpm for 15 min at 4°C. Conditioned media were collected every 24h during osteoclast formation and then tested by Enzyme-linked immunosorbent assay (ELISA, R&D Systems Inc., USA) to confirm that preosteoclasts release PDGF-BB. Conditioned medium from preosteoclasts (PO-CM) with serum were collected for co-culturing with EPCs and MSCs. Serum-free PO-CM were collected for migration experiments. After the preosteoclasts being treated by different concentrations of ZOL for two days and washed by PBS twice, the ZOL-free conditioned medium from preosteoclasts (PZ-CM) were collected and tested by ELISA for PDGF-BB, as well. Besides, conditioned medium from preosteoclasts added with the neutralizing antibodies for PDGF-BB (ab23914, polyclonal, Abcam, 1 μg/ml, USA) for two hours before collection (PA-CM). The PA-CM were collected for negative control to make sure the intermediary function of PDGF-BB and conditioned media from macrophages/OPCs (M-CM) were also obtained as negative control in parallel and applying the same protocol, respectively.

After de-paraffinization and rehydration of the tissue sections, they were boiled for 10 min in 0.1 M citrate buffer (pH 6.0). Next, sections were placed for 2 h in 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) and incubated at 4 °C with the primary antibodies anti-anti-IL-6, TNF-α, IL-10 anti-TGF-β1, anti-VEGF-A, anti-PDGF-B, collagen IV and CD31 overnight (Cat. No. ab9324, ab220210, ab33471, ab215715, ab1316, ab23914, ab6586 and ab182981, respectively, ABCAM, Cambridge, UK), all at 1 μg/mL. Anti-mouse or anti-rabbit HRP-DAB cell and tissue staining kits (Cat. No. CTS002 and CTS005, R&D Systems, Minneapolis, MN, USA) were used. After mounting, photographs of the slides were taken using a light microscope with a digital camera (Nikon SMZ 1000 and Nikon DS-Fi1, Tokyo, Japan). ImageJ software was used for image analysis (1.52a, NIH, Rockville, MD, USA). At least 3 sections per rat were included.

Cross-sections were obtained from paraffin-embedded femoral arteries and then incubated with anti-F4/80 antibody (Abcam Japan, Chuo, Tokyo, Japan; Ab204467; RRID: AB_2810932; raised in rat; 1: 50) and anti-PDGF-B antibody (Abcam Japan, Ab23914; RRID: AB_2162180, raised in rabbit, 1:50) overnight. The antibodies were diluted with Antibody Diluent (S3022; Dako, Santa Clara, CA, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (D1306; Thermo Fisher Scientific, Waltham, MA USA). The immunofluorescence images were obtained by a confocal microscope (BZ-X710 microscope; Keyence, Osaka, Osaka, Japan).

The FLX, WGO, alpha-Cyclodextrin (α-CD), and ELISA kit for hydroxyproline were supplied by R&D Systems, Inc. a Bio-Techne Brand (Minneapolis, MN, USA). Antibodies for TNF-α ((TNFA/1172); ab220210), vascular endothelial growth factor A (VEGF-A) (ab231260), PDGF-B (ab23914), and TGF-β1 ((EPR21143); ab215715) were purchased from Abcam (Cambridge, UK). When not otherwise specified, all other chemicals of the highest commercially analytical grade were supplied by Sigma-Aldrich (St. Louis, MO, USA) or Thermo Fisher Scientific Inc. (Pittsburgh, PA, USA).

Paraffin embedded sections were also stained with primary antibodies for PDGFB (ab23914, ABCAM, USA), HRAS (sc-29, SANTA CRUZ, USA), p53 (#2524, Cell Signaling Technology, USA), Ki67 (ab15580, ABCAM, USA), NQO1 (#3187, Cell Signaling Technology, USA), 4-HNE (MA5-27,570, Invitrogen, USA), Desmin (MA106401, Invitrogen, USA), CD31 (MCA1746GA, Invitrogen, USA), Collagen IV (ab6586, ABCAM, USA) with Opal Detection Kit (AKOYA BIOSCIENCES, USA) and the images were taken by Vectra Polaris Multispectral Imaging System (AKOYA BIOSCIENCES, USA) [29 (link), 30 (link)].

Interlaminar ligamentum flavum, interspinous ligament, or supraspinal ligament tissue was obtained from AS patients and traumatic injury patients who had spinal fractures when undergoing spinal surgery as controls. The tissues were fixed in 4% paraformaldehyde for 4–6 h and then embedded in paraffin. The paraformaldehyde-fixed paraffin-embedded sections were cut into 5 μm using a Leica RM2235. Paraffin-embedded sections were first deparaffinized twice in a series of 100% xylene, rehydrated in a series of graded ethanol (100%, 100%, 95%, and 80%), and then washed briefly in distilled water and PBS, respectively. Antigen thermal repair was performed with 0.01 M citrate buffer (pH6.0) or EDTA buffer (pH9.0) at high pressure, washed in PBS, sections were treated with 3% hydrogen peroxide-methanol for 10 min and blocked with normal goat serum for 30 min. Then, the sections were incubated overnight with PDGFB (1:100, Abcam, catalog: ab23914); GRB2 (1:50, Abcam, catalog: ab32037); P-ERK (1:100, Abcam, catalog: ab278538); RUNX2 (1:50, Abcam, catalog: ab76956) antibody at 4 °C. Goat anti-rabbit IgG secondary antibody (JACKSON, catalog: 111–035-003) was incubated and DAB solution (Sigma, catalog: D8001) was used for color development. All images were obtained using an Open-field slice scanner (NanoZoomer S210).