Ma5 14238

The concentrations of IL-1β and IL-6 in synovial fluid were analyzed using commercial ELISA kits (IL-1β, Cusabio, CSB-E06900Rb, Country; IL-6, RayBiotech, ELL-IL-6-1, USA) according to the instruction manual.
Fixed tissues were decalcified in commercial unbuffered 10% EDTA decalcification solution (Milestone, 51413G, USA) and embedded in paraffin. Paraffin-embedded tissue was sectioned to a thickness of 4 μm and stained with H&E or Safranin O according to standard protocols. The degree of OA progression was evaluated using the methods of Osteoarthritis Research Society International (OARSI) as described previously [21 (link), 22 (link)]. To perform immunohistochemistry (IHC), the sectioned specimens were stained with antibodies against type II collagen (Sigma-Aldrich, CP18-100UGCN, USA) and MMP13 (Invitrogen, MA5-14238, USA) according to standard protocols.

Samples were fixed in 10% neutral buffered formalin for 24 h and thereafter went through an ethanol dehydration series, xylene exchange and paraffin embedding following standard procedures, and sectioned at 6 µm thickness. For histology, sections were rehydrated and stained with hematoxylin (Harris cat #HHS16, Sigma-Aldrich) and eosin (alcoholic, Sigma-Aldrich, cat#HT110132) or Safranin O (Sigma 477-73-6) and Fast Green (Sigma 2353-45-9) to study cell morphology and sulfated proteoglycan deposition, respectively. For immunohistochemistry (IHC), sections were pre-incubated for 10 min with 3% H₂O₂ in methanol solution to quench endogenous peroxidase activity. Nonspecific binding was then suppressed with 1% horse serum (Vector Labs) in PBS for 45 min. Sections were then incubated overnight at 4 °C with primary antibodies against Collagen II (Invitrogen MA5-12789, 1:100), MMP13 (Invitrogen MA5-14238, 1:25) and ADAM-TS4 (Invitrogen MA5-26715, 1:150) followed by 30-min incubation with biotinylated secondary antibody (Vector Labs). Immunostaining was detected using horseradish peroxidase (HRP)-conjugated streptavidin and Vector® NovaRED™ peroxidase substrate, with hematoxylin (Vector Labs) as counterstain. After staining, both histology and IHC slides were dehydrated, mounted, coverslipped and imaged with microscope (Nikon Eclipse E800).

Multiplex IHC was used to analyze the co-localization of KIF14, Mieap, and EZR proteins in torpedo-like structures. Multiplex IHC was performed with a Bond RXm system (Leica, Hamburg, Germany) with antibodies against KIF14 (HPA038061, 1:500, Sigma, St. Louis, MO, USA; detected by Opal 520), Mieap/SPATA18 (HPA036854, 1:100, Sigma, St. Louis, MO, USA; Opal 620), EZR (HPA021616, 1:1000, Sigma, St. Louis, MO, USA; Opal 690), and MMP13 (MA5-14238, 1:25, Thermo Fisher Scientific, Waltham, MA, USA). Protein blocking was performed using 3% BSA-PBS (Sigma, St. Louis, MO, USA). TSA visualization was performed with the Opal 520, Opal 620, and Opal 690 (Opal seven-color IHC kit, Perkin Elmer, Waltham, MA, USA). Staining was finished with a DAPI counterstain and slides were enclosed in fluorescence mounting medium (Agilent, Santa-Clara, CA, USA). Slides were scanned using the Vectra 3.0 (PerkinElmer, Waltham, MA, USA). Tissue imaging was performed using inForm Advanced Image Analysis software (inForm 2.1.1 and 2.2.1; Perkin Elmer, Waltham, MA, USA).