Anti vegf antibody

Immunofluorescence staining was performed on sections of synovial tissues. Briefly, synovial tissues were obtained from patients with RA and OA. Cryosections (7 μm thick) were fixed with acetone for 15 min at room temperature, and blocked with 10% goat serum for 30 min at room temperature. After overnight incubation at 4°C with anti-human RANKL antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-VEGF antibody (R&D Systems), the samples were incubated with the secondary antibodies, anti-mouse FITC (Santa Cruz Biotechnology) and anti-rabbit PE (Southern Biotech, Birmingham, AL). The stained sections were visualized under a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) at ×200 and ×400 magnifications.

The tail tissues were fixed in 4% paraformaldehyde for 72 hours and then stored in 70% ethanol before µCT measurement or histological processing. A µCT35 desktop cone‐beam scanner (Scanco Medical) was used to scan the mouse tail at a resolution of 12 µm with a 55 kVp source and a 145 µAmp current. We performed three‐dimensional (3D) reconstruction on Co4‐Co7 vertebra. After CT reconstruction, we measured the intervertebral height of punctured disc normalized to adjacent vertebral body lengths using ImageJ. For comparison, the intervertebral heights of intact discs were also measured (n = 5).
After µCT examination, the tail tissues were decalcified with 10% formic acid for 3 weeks and then processed with a tissue processor (Excelsior™ AS Tissue Processor). The tail tissues were dehydrated with graded ethanol, immersed with xylene and embedded in paraffin. Serial sectioning was performed at thickness of 3 µm within the midsagittal region of the intervertebral disc. The sections were stained with Alcian blue & Orange G staining for histological analysis. Immunohistochemistry (IHC) was performed as previously reported,15 with sections incubated with 1:50 anti‐VEGF antibody (R&D Systems) or 1:200 anti‐Smad1 antibody (Abcam).