Af3715

The paraffin-embedded tissues were sectioned into 4-μm-thick sections. The sections were dewaxed, rehydrated and rinsed. The antigens were retrieved by heating the tissue sections at 100°C for 20 min in citrate (10 mmol/L, pH 6.0) solution when necessary. The sections were subsequently immersed in a 3% hydrogen peroxide solution for 10 min to block endogenous peroxidase activity and were incubated with the primary antibody sheep anti-human FAP (1:200, AF3715, R&D, USA) at 4°C overnight. A negative control was performed by replacing the primary antibody with PBS. The sections were then incubated with a horseradish peroxidase labeled secondary antibody (1:100, Boster, China) at room temperature for 120 min. Finally, the signal was developed for visualization with 3, 3′-diaminobenzidine tetrahydrochloride, and all of the slides were counterstained with hematoxylin.

FACs analysis of fibroblasts was performed using a panel of typical CAF markers; podoplanin (clone 8.1.1), PDGFRα (clone APA5) and β (clone APB5) (all 1:300, all BioLegend), FAP-α (1:50, AF3715; R&D Systems); and markers to exclude immune cells (MHCII; clone KH74 and CD45; clone 30-F11), epithelial cells (EpCAM, clone G8.8; Biolegend) and endothelial cells (CD31, clone 390; BioLegend). Morphological characteristics assed and visualised using an EVOS microscope and functional analysis was measured in terms of collagen gel contraction capacity. 1.5 × 10⁵ cells were seeded into 2 mg ml⁻¹ collagen gel (BD Biosciences) and detached from the sides of 24 well plates. Gel contraction was imaged over time and quantified as percentage area change over time. CAFs displayed typical CAF morphology, marker profiles and functionality were utilised (Supplementary Fig. 1).

Human synovium, cartilage or mouse knee joint samples were fixed in 4% paraformaldehyde overnight at 4 °C. Mouse knee joints were decalcified in 10% EDTA (pH 7.4) for 5–7 days. All samples were dehydrated in 30% sucrose for 1 day at room temperature and sectioned at 10 μm using the CryoJane tape-transfer system (Leica). Sections were incubated in blocking buffer (PBS with 5% horse serum) for 1 h at room temperature and then stained with anti-FAP (R&D, AF3715, 1:500), anti-OLN/CLEC11A (R&D, BAF1904, 1:500), anti-Oln/Clec11a (R&D systems, AF3729, 1:500), anti-beta galactosidase (GeneTex, GTX77365, 1:500) or anti-Col2a1 (Boster, BA0533, 1:200) antibodies in blocking buffer overnight at 4 °C. Slides were washed 3 times with PBS and then stained with Alexa Fluor 647 donkey anti-Sheep IgG (H+L) (Thermo Fisher Scientific, A21448, 1:500), Alexa Fluor Plus 555 donkey anti-goat IgG (H+L) (Thermo Fisher Scientific, A32816, 1:500) or Alexa Fluor 488 donkey anti-chicken IgY (IgG) (H+L) (Jackson ImmunoResearch, 703-545-155, 1:500) secondary antibodies in blocking buffer for 1 h at room temperature. Slides were then washed 3 times with PBS, stained with 1 μg/ml DAPI for 1 min and mounted with Anti-fade Prolong Gold (Invitrogen). Images were acquired using an Olympus IX73 microscope.

Whole protein extracts were obtained using RIPA buffer supplemented with Complete O, Mini, EDTA-free Protease Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany) and quantified using BCA Protein Assay kit (Pierce, Rockford, IL, USA). Proteins (40 µg) were electrophoretically separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked in Tris-Buffer Saline (TBS)-0.5% Tween 20 with 2% bovine serum albumin, and then incubated with the primary antibodies against proteins of interest at 4 °C overnight. Used antibodies (1:1000 dilutions) were: mouse monoclonal anti-human biglycan (BGN) antibody (MAB2667, R&D, Minneapolis, MN, USA), sheep polyclonal anti-human fibroblast activation protein alpha (FAP) antibody (-AF3715, R&D, Minneapolis, MN, USA), rabbit-monoclonal anti-human collagen type X alpha 1 (COL10A1) antibody (NBP2-66988, Novus Biologicals, Littleton, CO, USA), and anti-beta actin monoclonal antibody (AC-15) (AM4302, Invitrogen, Carlsbad, CA, USA). Secondary antibodies used were: anti-mouse (HAF007, R&D, Minneapolis, MN, USA), anti-rabbit (HAF008, R&D, Minneapolis, MN, USA), and anti-sheep (HAF016, R&D, Minneapolis, MN, USA), all conjugated with HRP. Signals were developed using ECL HRP chemiluminescent substrate (WP20005, Invitrogen) and captured using MicroChemi 4.2 system (Bio Imaging Systems).

We incubated 4 μm thick, paraffin-embedded sections of mouse lesional skin with rat anti–procollagen I (1:100; clone M-58, MilliporeSigma) primary Abs, followed by species-appropriate secondary Abs conjugated to Alexa Fluor 594 (Invitrogen, Thermo Fisher Scientific, A21201). Nuclei were detected using DAPI. Healthy controls and SSc skin sections were incubated with sheep anti-FAP (1:100 dilution; catalog AF3715, R&D Systems, Bio-Techne) and rabbit anti–Thy-1 (1:50 dilution; catalog ab92574, Abcam) primary Abs, followed by species-appropriate secondary Abs conjugated to Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, A21200) or 647 (Jackson Immunoresearch, 313-607-003). Nuclei were detected using Hoechst 33342 (NucBlue Live Ready Probes, Molecular Probes, Thermo Fisher Scientific). Slides were evaluated using a Nikon A1 laser scanning confocal microscope.

Total protein was extracted using a lysis buffer and protease inhibitor (Beyotime Biotechnology, China). Equivalent protein amounts were denatured in an SDS sample buffer, and then were separated by SDS-PAGE and transferred onto polyvinylidene difluoride mem-brane. After being blocked with 5% non-fat dry milk in PBS containing 0.05% Tween-20, the blotted membranes were incubated with anti-human FAPα antibody (1:2000, AF3715, R&D, USA) and secondary antibody (1:5000, Boster, China) thereafter. GAPDH protein levels were also determined by using the specific antibody (1:1000, Boster, China) as a loading control.