S1699

To evaluate the ratio of TMEM119⁺ cells (microglia) to Iba-1⁺ cells (microglia and macrophages), immunofluorescence staining was performed. After deparaffinization and rehydration, heat-induced antigen retrieval using a target retrieval solution (pH 6.1; S1699, Agilent Technologies, California, USA) was performed. Next, the sections were incubated in a serum-free protein block (Agilent Technologies, California, USA) at T_A for 30 min and then incubated with anti-Iba-1 antibody and anti-TMEM119 antibody at 4 °C overnight. Primary-stained sections were incubated with appropriate Alexa Fluor-conjugated secondary antibodies for 30 min at T_A. The following secondary antibodies were used, respectively: Donkey anti-goat IgG secondary antibody Alexa Fluor 488 (Invitrogen, Massachusetts, USA) and Donkey anti-rabbit IgG secondary antibody Alexa Fluor 568 (Invitrogen, Massachusetts, USA). After washing with PBS, sections were mounted in Vectashield mounting medium with DAPI (Vector Laboratories, California, USA). Images of specimens were obtained by fluorescence microscopy (BZ9000, Keyence, Osaka, Japan).

IHC staining was performed to detect FZD2 expression in tissue samples. The standard streptavidin–biotin–peroxidase complex method was used for IHC staining. Briefly, after blocking of endogenous peroxidase activity in tissue sections with 3% H₂O₂ and antigen retrieval with a target retrieval solution (S1699; Agilent Technologies Inc., Santa Clara, CA, USA) according to the manufacturer's instructions, the sections were incubated with 10% normal goat serum (S-1000; Vector Laboratories, Burlingame, CA, USA) in PBS for 30 min. Next, sections were incubated with a primary anti-FZD2 antibody (ab109094, 1:200, Abcam, Cambridge, UK) at 4°C overnight. After three washes with PBS, the sections were incubated with donkey anti-goat IgG H&L (HRP) (ab205723, 1:2000, Abcam, Cambridge, UK) for 30 min at room temperature. Finally, sections were incubated with a peroxidase substrate solution (Sk-4100, Vector Laboratories, Burlingame, CA, USA) until the desired staining intensity was attained. Sections were rinsed with tap water, counterstained with haematoxylin, and mounted with coverslips. The results of IHC staining were viewed and scored separately by two experienced pathologists. The expression levels of FZD2 expression were assessed and scored as follows: negative (0; complete absence of staining), weak staining (score: 1), moderate staining (score: 2), or strong staining (score: 3).

The 3 µm thick paraffin sections were subjected to deparaffinization/hydration as follows: (1) suspension in xylene for 10 min for four times; (2) 5 min in isopropanol twice; (3) 5 min in 96% ethanol twice; (4) 5 min in 70% ethanol; (5) 10 min in double distilled water twice. Antigen unmasking was performed by boiling slides in a citrate-based target retrieval solution (Agilent Dako #S1699, Jena, Germany) for 20 min. Then, the slides were cooled to room temperature and the sections were washed in PBS twice, followed by incubation in 0.1% Triton X-100-PBS for 4 min. The slides were washed with PBS two more times. The slides were blocked for 45 min using an immunoblock solution (Roth #T144.1, Karlsruhe, Germany). The primary antibody against CD3 (clone M-20, Santa Cruz Biotechnology, #sc-1127) or FoxP3 (clone 2A11G9, Santa Cruz Biotechnology, #sc-53876, Dallas, TX, USA) diluted 1:50 in the immunoblock solution was added to the slides and was allowed to incubate for ≥16 h at 4 °C. Signal detection was obtained using Histofine Simple Stain Max PO detection system (#414161F, Nichirei Biosciences Inc., Tokyo, Japan) and DAB peroxidase substrate kit (#SK-4100, Linaris, Vector laboratories, Dossenheim, Germany). Counterstaining was employed using hematoxylin. Finally, images were acquired on a Keyence microscope (BZ-8000K, Osaka, Japan).

The knee joints were fixed in 10% neutral buffered formalin at room temperature for 24 h then decalcified in Immunocal (StatLab, McKinney, TX) for 3 days; fresh Immunocal was changed every 24 h. Tissues were processed and paraffin-embedded, then 5-μm-thick sagittal sections were generated, starting from the medial side of the knees. They were stained with Safranin-O. OARSI scoring was based on an established scoring system [39 (link)]. Synovitis scoring was based on the severity of synovium hyperplasia (score range 0–3) and inflammatory cell infiltration (score range 0–3) in the sub-synovial region [40 (link)]. All histological scoring was performed by two blinded reviewers.
For immunohistochemistry, the sections were deparaffinized and rehydrated using xylene followed by an ethanol gradient. Antigen retrieval was performed using citrate buffer (Dako, S1699) at 3 psi. The sections were incubated overnight at 4 °C with primary antibodies against GSDMD (1:200, Abcam, ab219800), followed by 1 h incubation with biotinylated goat secondary antibody (Vector Laboratories, BA-9200). DAB reagent (Vector Laboratories, SK-4100) was used for the peroxidase-substrate reaction.

The paraffin-embedded samples were cut into 4 µm sections, deparaffinized, and incubated in 10 mM citrate buffer (pH 6.0, S1699; Dako, Glostrup, Denmark) for 20 min in a microwave oven. Blocking buffer (Dako) with 10% fetal bovine serum in PBS was added to the slides for 10–20 min, and then a specific serum-free protein block (Dako) was added for 30 min at RT. After washing, the sections were stained using Dako Autostainer Plus [16 (link)] with the following primary antibodies: rat monoclonal anti-CD66b antibody (1:500; ab233811, clone 2F12E8; Abcam, Cambridge, United Kingdom), rabbit monoclonal anti-BDNF (CRC patients 1:500, for mice 1:1000, pH 6, RT, 1 h; ab108319, Abcam). The secondary antibody Envision+ System-HRP-labelled polymeric anti-rat/anti-rabbit antibody (Dako) was visualized using 3,3′-diaminobenzidine (DAB) substrate (Vector Laboratories Inc., Burlingame, CA, USA) and counterstained with hematoxylin.