Af4488

For immunofluorescence staining, rehydrated sections were immersed in a solution of proteinase K (20 mg/ml; Thermo Fisher Scientific) in 95% (v/v) TE buffer [50 mM tris-HCl, 1 mM EDTA, and 0.5% (v/v) Triton X-100 (pH 8)] with 5% (v/v) glycerol and incubated for 15 min at 37°C. Sections were washed with PBS and permeabilized using 0.1% Triton X-100 in PBS for 10 min at RT. The sections were immersed in a blocking solution [1% (w/v) BSA, glycine (0.25 M), normal donkey serum (5%, v/v), and normal goat serum (5%, v/v) in tris-buffered solution (TBS)] and incubated for 1 hour at RT. Sections were then incubated with primary antibody against CD39 (5 μg/ml; AF4398, R&D Systems), CD73 (5 μg/ml; AF4488, R&D Systems), and A2BR (1:200; MBS8207549, MyBioSource, San Diego, CA) in diluent solution (1%, w/v) and normal donkey serum (1%, v/v) in TBS overnight at 4°C. Sections were stained with secondary antibody using anti-donkey or anti-goat Alexa Fluor 647 (1:250; Jackson ImmunoResearch, West Grove, PA). For immunocytochemical costaining, cells were incubated with primary antibody against CD39 (1:100; ab227840, Abcam, Cambridge, UK) and CD73 (5 μg/ml; AF4488, R&D Systems) and stained with secondary antibody using anti-donkey Alexa Fluor 488 and anti-goat Alexa Fluor 647 (1:250; Jackson ImmunoResearch). Images were acquired and presented as pseudocolors.