Adenosine kinase (AK) expression during hypoxia in vivo. (A) C57BL/6 mice were exposed to ambient hypoxia (8% oxygen) or normoxia for 4 hours. The colon (Co), lungs (Lg), liver (Lv), muscle (Mu), heart (Ht), kidneys (Kd), and brain (Br) were harvested and AK or CD73 transcript was determined relative to β-actin by real-time RT-PCR (*P < .05). (B) Western-blot analysis of AK or VEGF protein from kidney, liver, or colon. (C) Normoxic or hypoxic cardiac tissue was harvested and stained with AK antibody. Note attenuated AK staining in hypoxic vasculature (magnification, ×400). IgG controls were used at identical concentrations and staining conditions as the target primary antibodies for AK. (D) The tissue was fixed in Tissue-Tek (Sakura) and cut in 3-μm slices, mounted on glass sildes, air dried, post-fixed in chilled methanol for 10 minutes, and permeabilized in chilled acetone for 1 minute. Immunohisto-chemical stainings were performed with polyclonal rabbit anti-AK antibody (dilution 1:100; Santa Cruz Biotechnology, Santa Cruz, CA) for AK and rabbit IgG serum (1:100; Santa Cruz Biotechnology) for isotype control. First endogneous peroxidase activity was quenched by incubating the specimens for 30 minutes with DAKO peroxidase blocking buffer (DAKO, Hamburg, Germany). Primary antibodies were applied for 30 minutes. Incubation with the DAKO evision secondary antibody followed for another 30 minutes. Staining was completed by 10-minute incubation with 3,3′-diaminobenzidine (DAB)+ substrate chromogen, which results in a brown-colored precipitate at the antigen side, and a hematoxylin counterstaining for 2 minutes. Evaluation of the immunohistochemical staining was performed using a LEITZ DMRBE light microscope (40×/0.75 lens), Leica DC300F camera, and Leica QWin software (Leica, Heidelberg, Germany).