Antigen decloaker solution

For APP immunohistochemistry staining, antigen retrieval was performed on four transverse 40 µm free floating sections, ∼200 µm apart spanning from ∼ −3.0 mm to −4.5 mm relative to bregma, with Antigen Decloaker solution (Biocare Medical, Concord, CA, USA) before sections were placed in 2% H₂O₂ for 20 min and blocked with 3% normal goat serum in Tris-buffered saline (TBS) containing 0.25% Triton X (TBS-X) for 1 hr. Sections were incubated overnight in TBS-X solution containing rabbit polyclonal beta-amyloid antibody (1:2000, Thermo-Fisher Scientific) and 1.5% normal goat serum; 24 hr later, sections were rinsed with TBS and incubated for 1 hr with anti-rabbit HRP-conjugated secondary antibody (ABC Elite kit, PK6100, Vector Laboratories, Burlingame, CA). Following incubation, sections were rinsed via TBS and stained with diaminobenzidine for visualization (diaminobenzidine, Vector Laboratories, Burlingame, CA).

After the MRI examination, rats were sacrificed by intraperitoneal administration of overdose pentobarbital sodium. The samples were fixed with 4% paraformaldehyde for 48 h, decalcified in 10% EDTA, embedded in paraffin, and sectioned (5 μm) along the midsagittal plane. Sections were used for hematoxylin–eosin (H&E) and safranin-O/fast green staining. For Immunofluorescent analysis, antigen retrieval was performed using Antigen-decloaker solution (Biocare Medical, Walnut Creek, CA, USA). Slides were incubated with antibodies targeting collagen-II, aggrecan, MMP-13, and ADAMTS-5, respectively. Histological images were analyzed under the BX53 microscope (Olympus). The grading score after histological staining was obtained by the criteria established by Masuda^{43 (link)}. All image assessments were performed by 2 independent blinded observers.