Harris modified hematoxylin

Samples were dewaxed and rehydrated as per immunohistochemical protocol described above. For H&E, a regressive hematoxylin (Harris’ modified hematoxylin, Sigma, Cat.HHS128) and Eosin Y (Sigma, Cat.230251) protocol was performed. In brief, slides were submerged in hematoxylin for 80 sec, followed by a 3 sec submersion into 1% acid alcohol solution, and 2 min incubation in Scott’s tap water (pH8.0). For Sirius Red, samples were submerged in Picro’s Sirius Red (Abcam, Cat.ab150681) for 1 h, followed by a 1 min 0.1 N HCl wash. Slides were dehydrated and mounted using DPX.

To confirm FCM data, a CD45 and HLA-DR double staining was performed on OA-IFPs tissue sections from samples used for stem cells isolation.
Osteoarthritic infrapatellar fat pad specimens were fixed in 10% formalin in PBS, paraffin-embedded and cut into 4 μm-thick sections. After deparaffinization, the sections were treated with EDTA (pH = 8; for Mouse Anti-Human-CD45) and citrate buffer (pH = 6; for Mouse Anti-Human HLA-DR) in microwave oven heating. After washing in PBS, the specimens were incubated with Dual Endogenous Enzyme block (ready to use – Dako, Milan, Italy) to assure endogenous peroxidase and alkaline phosphatase inhibition, rinsed in PBS, blocked in 0.2% BSA (Bovine Serum Albumin) and incubated with Mouse Anti-Human CD45 1:100 (Biocare CM016) overnight at 4°C. After washing, the samples were incubated with the secondary antibody (HRP-conjugated Goat Anti-Mouse – Jackson ImmunoResearch, Cambridgeshire, United Kingdom) and the reaction was detected with diaminobenzidine, Dako (brown stain). Thus, the sections were rinsed in PBS and incubated with Mouse Anti-Human HLA-DR 1:100 (Clone TAL.1B5 – Dako) overnight at 4°C. The slides were subsequently rinsed in PBS, covered with Mouse AP Polymer and HLA-DR was visualized with GBI-Permanent Red solution (Red stain). The samples were counterstained with Harris modified hematoxylin (Sigma, Milan, Italy) before mounting.

Trifluoroacetic acid, Harris-modified hematoxylin, and α-cyano-4-hydroxycinnamic acid (CHCA) were obtained from Sigma-Aldrich. High-performance liquid chromatography (HPLC) grade methanol, ethanol, aceto-nitrile, xylene, hydrogen peroxide, and water were obtained from Thermo Fisher Scientific. Recombinant peptide N-glycosidase F (PNGase F) PRIME and endoglycosidase F3 Prime (Endo F3) were obtained from N-Zyme Scientifics.

Tissue sections were dewaxed, rehydrated and treated sequentially with Avidin/Biotin Blocking Kit (Abcam), 3% H₂O₂ and normal swine serum (Reactolab, Servion, Switzerland) to reduce non-specific staining. Sections were then incubated for 1 h at 37°C with either polyclonal rabbit anti-HTRA1 (1:300), polyclonal rabbit anti-HTRA3 (1:400), or equivalent dilutions of normal serum. After washing in PBS, sections were incubated with a biotinylated swine anti-rabbit IgG (1:400) for 45 min at 37°C followed by washing and a further incubation for 30 min with Vectastain (Reactolab). Sections were developed using 3,3' diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich), counterstained with Harris modified hematoxylin (Sigma-Aldrich) and visualized using an Olympus BX51 light microscope (Olympus Schweiz AG, Volketswil, Switzerland).