Target retrieval solution 10

Following deparaffinization and dehydration, tissue samples were washed in PBS. Sections were incubated at 95–100 °C for 30 min in Target Retrieval solution 10× (Dako, Santa Clara, CA, USA) for antigen retrieval, then incubated with 3% H₂O₂ to block intrinsic peroxidase activity. Non-specific antibody-binding sites were blocked using 3% normal rabbit serum (Nichirei, Tokyo, Japan). Specimens were incubated with appropriately diluted primary mouse anti-F4/80 and anti-CD4 antibodies at 4 °C overnight. After three PBS washes, specimens were incubated in the secondary antibody (biotin-labelled rabbit anti-rat IgG; Vector Labs, Burlingame, CA, USA) for 60 min at 20 °C. Staining was detected with 3,3′-diaminobenzidine (DAB; Nichirei) and haematoxylin was used for counterstaining. Slides were observed under a light microscope (SZ61; Olympus)⁴² . In this experiment, 24 mice were assessed. Six mice with inflammatory stimulation and no intra-articular injection were regarded as the before-treatment group. Eighteen mice with inflammatory stimulation that underwent treatments were divided as follows: no treatment group (n = 6, PBS injection), single-cell treatment group (n = 6, ADSC single-cell injection), and spheroid treatment group (n = 6, ADSC spheroid injection).