α sm actin

Human and mouse sections from formaldehyde-fixed and paraffin-embedded lung tissues were deparaffinized and rehydrated. Epitope retrieval was performed by boiling the sections in citrate buffer, pH 6.0. Sections were reacted with hydrogen peroxide to block endogenous peroxidase, washed, and blocked with 5% goat serum. The sections were then incubated with the primary antibodies against GM-CSF (1:400; Abcam), TNF (1:400; Abcam), α-SM actin (1:1; Dako), CD31 (1:50; Dako), CD34 (1:1; Dako), CD68 (1:100; Dako), GM-CSFRα (1:50; Santa Cruz Biotechnology, Inc.), and Mac3 (1:200; BD) overnight at 4°C. After streptavidin-biotin amplification (LSAB2 kit [Dako] or Vectastain Elite ABC kit [Vector Laboratories]), the slides were incubated with 3, 3′-diaminobenzidine and counterstained with hematoxylin. A negative control was performed using mouse IgG or rabbit Ig instead of the primary antibody. The localization and intensity (0, 1+, 2+, 3+) of immunoreactivity were assessed by two independent examiners, blinded to the diagnosis of PAH or to the mouse treatment group. In the murine experiments described below, the number of Mac-3–positive cells per PA was counted for all vessels in the section and a mean value calculated per mouse.