Goat serum

Serial frontal sections of the paraffin-embedded samples were cut at a thickness of 6-µm, and were mounted on silane-coated glass slides (Muto). The sections were de-waxed in xylene and re-hydrated in descending concentrations of ethanol. The slides were treated with antigen retrieval buffer (10 mM sodium citrate buffer, pH6.0) and microwaved for 5 min, and 10% goat serum (Nichirei Bioscience, Tokyo, Japan) was used to block non-specific immunoreactions. The specimens were incubated with the primary antibody, rabbit polyclonal anti-Itm2a antibody (1∶1000, 18306-1-AP; Proteintech Group, Inc., Chicago, IL) at 4°C. The primary antibodies against Itm2a were visualized using an Alexa Fluor 568-conjugated anti-rabbit secondary antibody (1∶2000, A11011; Invitrogen, Carlsbad, CA). Finally, the immunostained sections were counterstained with 4′6-diamidino-2-phenylindole (DAPI, Dojindo, Kumamoto, Japan). The sections were rinsed in PBS three times at every interval between the steps.
The immunoreactivity of hepatocytes (Fig. S2) and chondrocytes was used as a positive control to develop the immunohistochemical staining procedures based on the manufacturer’s instructions and previous studies [18] (link), [20] (link), [31] (link).

Cells were fixed with 4% paraformaldehyde (Wako) for 15 min, permeabilized with 0.1% Triton X-100 for 5 min and blocked with 10% goat serum (Nichirei Bioscience) in PBS for 1 hr at room temperature (RT). Cells were incubated with primary antibodies for 2 hr at RT before incubation with appropriate secondary antibodies for 1 hr at RT, prior to counterstaining with 4’,6-diamidino-2-phenylindole (DAPI)(Dojindo Laboratories). Samples were observed using a Leica fluorescent microscope (Leica Microsystems). The primary antibodies used were: anti-nestin antibody (1:100; Millipore), anti-fibronectin antibody (1:100; Sigma-Aldrich), anti-αSMA antibody (1:100; Sigma-Aldrich), anti-S100β antibody (1:100; Sigma-Aldrich) and anti-trichohyalin antibody (1:5; Santa Cruz Biotechnology). Secondary antibodies used were: Alexa Fluor 488-conjugated goat anti-mouse (1:200) and Alexa Fluor 546-conjugated goat anti-rabbit (1:200, both from Life Technologies).

Histological analyses were carried out as described previously (Niimi et al. 2018 (link); Zhu et al. 2017 (link)) with some modifications. The sections were washed with water after deparaffinization in xylene and dehydration in ethanol. The sections were incubated in 3% H₂O₂ diluted with phosphate buffered saline (PBS) for 15 min to remove endogenous peroxidase activity and incubated in 10 mM citrate buffer for 60 min at 90 °C for antigen retrieval. After three 5-min PBS washes, the sections were blocked in 10% goat serum (#426041, Nichirei Bioscience, Tokyo, Japan) for 10 min and incubated with a polyclonal rabbit anti-VDAC primary antibody (ab15895, Abcam, Cambridge, UK) at a dilution of 1:3000 overnight at 4 °C. Normal rabbit IgG (#2729, Cell Signaling, Beverly, MA, USA) diluted to the same concentration as a primary antibody was used as the negative control. After being washed three times for 5 min in PBS, the sections were incubated in Histofine Simple Stain Max PO (#414181, Nichirei Bioscience) as a secondary antibody at room temperature for 30 min. After three 5-min washes in PBS, the signals were developed using a diaminobenzidine (DAB) substrate solution kit (#425011, Nichirei Bioscience) and counterstained with hematoxylin.

To determine the localization of phosphorylated TAK1 (p-TAK1) and TNF-α, the paraformaldehyde-fixed synovial tissue samples were embedded in paraffin and sliced into 3-μm thick sections. The sections were deparaffinized with xylene for 1 h, hydrated in serial dilutions of ethanol (100%, 95%, and 70%) and then rinsed in distilled water. For antigen retrieval, deparaffinized sections were immersed in 10 mM sodium citrate buffer pH 6.0 and maintained at 98 °C for 30 min. After cooling at room temperature, endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 15 min. The slides were washed in phosphate-buffered saline (PBS) and incubated with 10% goat serum (Nichirei, Tokyo, Japan) at room temperature. Subsequently, the sections were incubated overnight at 4 °C with rabbit polyclonal primary antibody against p-TAK1 (Thr¹⁸⁴/¹⁸⁷) (cat.no. #4531, Cell Signaling Technology Japan, Tokyo, Japan). After washings twice in PBS, the sections were incubated for 10 min at room temperature with biotinylated anti-rabbit IgG (Nichirei). Subsequently, the sections were washed twice in PBS and incubated with horseradish peroxidase (HRP)-conjugated streptavidin for 5 min. Peroxidase activity was revealed by 3,3′-diaminobenzidine (Nichirei) and the sections were counterstained with Mayer’s hematoxylin.