Immunostaining with monoclonal antibody to macrophages-2 (MoMa-2)

Aortic root sections were fixed for 15 minutes in 10% methanol-free formalin and washed three times with PBS. Sections were then blocked for 30 minutes to protect against endogenous tissue peroxidase activity (Signet Covance, Prineceton, NJ), washed twice with PBS, and then blocked with Ultra V protein block (Lab Vision, Fremont, CA) for 7 minutes. The sections were next incubated in Ultra V protein block solution (1:4 in PBS) containing monoclonal antibody to macrophages-2 (MoMa-2, 1:600; Acris, Hiddenhausen, Germany) for 1 hour at RT, and then overnight at 4°C.

The sections were next washed twice with PBS, incubated for 3 hours with an HRP-coupled polyclonal rabbit anti-rat secondary antibody (1:100, Dako Denmark A/S, Glostrup, Denmark), and washed three times with PBS. AEC substrate (Vector Laboratories, Burlingame, CA) was added until reaction development and then washed away 3 times with H₂O. Sections were counterstained with hematoxylin for 5 min, washed in 0.1% NaHCO₃, and mounted with glycerol-gelatin aqueous slide mounting medium (Sigma-Aldrich, St. Louis, MO). Images were taken with a Leica DM 4000B microscope equipped with a Leica DFC 500 camera. The intensity and extent of MoMa-2 staining of three consecutive sections per mouse was quantified using ImageJ software.