Ab124780

The collected hippocampus tissue was fixed in 4% paraformaldehyde solution overnight and then sliced into sections with a thickness of 5 μm, followed by embedding in paraffin for staining. The paraffin sections were stained with anti-A1 adenosine receptor antibody (ab124780, 1:1,000, Abcam, UK) at 4°C overnight and then stained using horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab205718, 1:2,000, Abcam, UK) at room temperature and a 3,3′-diaminobenzidine kit for 15 min. The nuclei were counterstained using hematoxylin (H3136, Sigma-Aldrich, USA), and the positive cells in five randomly chosen fields were counted and observed with a stereomicroscope. Here, the sample size was n = 10.

In order to detect CD39 and CD73 co-expression by immunocytochemistry, fixed synovial cells were stained for these cell surface ectonucleotidases using the primary antibodies rabbit anti-CD39 (ab178572, Abcam, Cambridge, UK) and mouse anti-CD73 (ab133582, Abcam, Cambridge, UK). The expression of ADA, ENT1, ENT2, and ARs was investigated by immunohistochemical staining of synovial tissue samples using the primary antibodies rabbit anti-ADA (NBP1-90361, Novus Biologicals, Cambridge, UK), rabbit anti-ENT1 (NBP1-84838, Novus Biologicals, Cambridge, UK), rabbit anti-ENT2 (ab48595, Abcam, Cambridge, UK), rabbit anti-A₁AR (ab124780, Abcam, Cambridge, UK), rabbit anti-A_2AAR (ab260032, Abcam, Cambridge, UK), rabbit anti-A_2BAR (LS-C20310, LSBio, via Biozol, Eching, Germany), and rabbit anti-A₃AR (LS-A686, LSBio, via Biozol, Eching, Germany). After blocking (10% bovine serum albumine, 10% chicken serum, and 10% goat serum), cytospin or tissue slides were incubated with primary antibodies overnight at 4 °C. Primary staining was visualized using Alexa Fluor-labeled secondary antibodies (goat anti-rabbit Alexa Fluor 594 or goat anti-mouse or anti-rabbit Alexa Fluor 488; Thermo Fisher/Life technologies, Schwerte, Germany). Cell nuclei were counterstained with DAPI (Merck, Darmstadt, Germany). Slides without primary antibody served as negative controls (Supplementary Materials Figure S2).

The experimental process of western blot was carried out as described previously³² with modification. The samples of the hippocampus were homogenized in a lysis buffer solution. Sodium dodecyl sulfate (SDS) polyacrylamide gel was used to separate proteins (30 μg/lane). Subsequently, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (0.22 μm, Millipore Corp). After soaking in 8% milk for 90 min, membranes were incubated with anti‐ADK (polyclonal rabbit, 1:200; from Professor Detlev Boison, Department of Neurosurgery, Robert Wood Johnson & New Jersey Medical Schools, Rutgers University) or anti‐A₁R (monoclonal rabbit ab124780, 1:100, Abcam).
Anti‐β‐actin (monoclonal mouse, 1:1000; Cell Signaling Technology) was used to normalize results. After incubating with HRP‐conjugated secondary antibodies (1:5000; Applygen Technologies Inc.), proteins were visualized using enhanced chemiluminescence reagents (Applygen, Technologies Inc.). Autoradiography films were scanned with a luminescent image analyzer (LAS‐3000; Fujifilm). The quantitative analysis process of protein strips was performed using ImageJ software.