Ab109231

Immunostaining was performed as previously described [16 (link)]. OA synovial tissues were used to determine protein expression of Axl, Mer, and Tyro3. Sections of synovial tissues were incubated with rabbit anti-human Axl (1:600; C89E7; Cell Signaling, Danvers, MA, USA), rabbit anti-human Mer (1:2000; ab52968; Abcam, Cambridge, UK), rabbit anti-human Tyro3 (1:500; ab109231; Abcam), or rabbit anti-human IgG (1:74,000; X0936; Agilent Technologies, Santa Clara, CA, USA) overnight at 4 °C and subsequently with biotinylated goat anti-rabbit IgG (1:400; PK-6101; Vector Laboratories, Peterborough, UK) for 30 min at RT. A biotin–streptavidin horseradish peroxidase detection system was used according to the manufacturer’s protocol (PK6101; Vector Laboratories). Bound complexes were visualized with diaminobenzidine by incubation for 10 min at RT. Sections were counterstained with hematoxylin. Pictures were taken with a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) at 20× and 40× magnification.

Gas6, Mer, Axl, and Tyro-3 proteins in brain tissues were measured by immunohistochemistry of 5-μm tissue sections. Anti-Myelin Basic Protein antibody (MBP antibody sc-271524, 1:500, Santa Cruz Biotechnology, Dallas, TX, United States) labels myelin which is the most abundant protein in the myelin membrane. For this, sections were deparaffinized, rehydrated, and treated with 3% (v/v) H₂O₂ in methanol for 30 min, followed by blocking with 5% (w/v) fat-free milk for 1 h. Subsequently, they were incubated with a primary antibody (anti-Mer, ab79223; anti-Axl, ab219651; or anti-Tyro 3, ab109231; anti-FOXP3 antibody, ab20034, 1:100, Abcam, UK) at 4°C overnight. After washing, bound antibodies were detected with biotin-labeled secondary antibodies using the ABC kit, visualized with diaminobenzidine, and examined by light microscopy (Thermo Fisher Scientific, USA). Immunostained slides were independently scored by at least three investigators and the mean of the results was calculated.