Ab58703

Western blotting was performed with primary antibodies against 14-3-3β/α (#sc25276; 1:500; Santa Cruz, CA, USA), haptoglobin (#ab256454; 1:1000; Abcam, Cambridge, UK), S100A6 (#ab181975; 1:10,000; Abcam), ClpP (#sc271284; 1:1000; Santa Cruz), A2M (#ab58703; 1:1000; Abcam), BVR-A (#sc393385; 1:1000; Santa Cruz), and β-actin (#C1313-100 1:1000; Applygen, Beijing, China) in tissues from the hippocampus, prefrontal cortex, and temporal lobe. A horseradish peroxidase conjugated labeled secondary antibody was used. Immunoreactivity was visualized by scanning the membranes with an Odyssey infrared imaging system (LI-COR). All procedures were performed by experienced experimenters blinded to the treatment condition.

The knee joints from mice were fixed with 4% paraformaldehyde for 24 h and then decalcified with 0.5 M ethylenediaminetetraacetic acid (EDTA) at pH 7.4 for 21 days. The specimens were embedded in paraffin and sectioned at 4 μm. For histological analysis, the samples were stained with hematoxylin and eosin (H&E) and safranin O-Fast Green staining (Sigma-Aldrich). For the immunohistochemistry analysis, we employed the following primary antibodies: A₂M (ab58703, Abcam), MMP13 (ab84594, Abcam), and Cleaved Caspase-3 (#9661, CST). Sections were then stained with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). For immunofluorescence, the primary antibodies used were Collagen II (ab34712, Abcam), Aggrecan (ab3773, Abcam) and Alexa 594 dye-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The sections were mounted with medium containing DAPI and images were obtained using a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan).

Sections were suffered as follows: dewaxed, hydrated, and blocked with 10% goat serum for 1 hour. Sections were then incubated with α2M (ab58703, Abcam, Cambridge, MA, USA) primaryantibody overnight at 4°C. After incubated with biotinylated IgG and Elite ABC reagent (Beyotime, Shanghai, China), sections finally were developed by 3, 3-diaminobenzidine and counterstained with hematoxylin.