Ab19345

At first, sections were deparaffinized by dimethylbenzene and rehydrated in descending series of ethanol. Then, the slides were incubated with 3% H₂O₂ at room temperature to eliminate endogenous peroxidase. Antigens were retrieved by maintaining the temperature between 92°C and 98°C in a 0.01 M citrate buffer (pH 6.0) for 4 mins. Subsequently, slides were then incubated with 10% (vol/vol) normal goat serum for 20-30 mins at room temperature in order to reduce nonspecific reactions.
Then, the slides were added with primary rabbit anti-VCAN polyclonal antibody (ab19345, Abcam, Massachusetts, US) with a 1 : 300 dilution in phosphate-buffered saline at 4°C overnight. The next day, the slides were incubated with biotin-labeled secondary antibody followed by horseradish peroxidase-linked antibody (Zymed, San Francisco, CA) for 30 mins at room temperature after rinsing with phosphate-buffered saline (PBS). Then, the sections were stained with 3,3-diaminobenzidine, and the nuclear was counterstained by hematoxylin. After that, the slides were rehydrated in ascending series of ethanol and then covered by neutral resin.

Frozen brachiocephalic artery sections were stained with antibodies against CD68 (marker for macrophages, MCA1957, AbD Serotec, UK), ADAMTS4 (a generous gift from Prof. Hideaki Nagase, Kennedy Institute of Rheumatology, Oxford University, UK), alpha smooth muscle actin (αSMA) (CGA7, Santa Cruz Biotechnology Inc., USA), ADAMTS4/1 cleaved versican V1 neoepitope DPEAEE (ab19345, Abcam, USA) or aggrecan ARGxx neoepitope (ab3773, Abcam, USA) with the corresponding secondary antibody conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotechnology Inc., USA) and visualized by applying 3,3′-diaminobenzidine tetrahydrochloride (DAB) or through Alexa fluor 488 or 568 (Life technologies, Singapore) and visualized under Axiovision compound microscope (Zeiss, Germany) or UltraView Vox Spinning Disk confocal microscopy (PerkinElmer, USA). Percentage positive staining was quantified using Adobe Photoshop CS8 software (USA). The vulnerability index was calculated as the ratio of (ORO stained area + CD68 positive area)/(αSMA area + collagen positive area). The specificity of the antibodies used is demonstrated using isotype antibody controls in all IHC/IF staining (Supplementary Fig. S9).