Example 3
An established murine model of coronary arteritis based on intraperitoneal injection of the cell wall extract of group B Lactobacillus casei (LCWE) was used. Lehman et al., 48 Clin. Immunol. Immunopathol. 108 (1988). Group B L. casei were grown and a cell wall extract (LCWE) was prepared as described. Schulte et al., 183 J. Immunol. 5311 (2009). Briefly, 6-week old C57/BL6 mice were injected with 250 μg of LCWE in phosphate buffered saline (PBS) or with saline alone. Fourteen days later, mice were sacrificed, and coronary arteries were identified in serial sections (7 μm), fixed with formalin, and stained with hematoxylin and eosin. For the immunohistochemical analysis, sections were pre-treated with 0.3% hydrogen peroxide in PBS for 30 min. Meprin A (clone F-20, Santa Cruz Biotech., Santa Cruz, Calif.) or isotype control antibody (goat serum, Santa Cruz Biotech.) was applied in 0.5% bovine serum albumin in PBS at 1:100 for 1 hr. Slides were then washed and biotinylated anti-goat horseradish peroxidase conjugated secondary antibody (Vector Lab, Burlingame, Calif.) was applied at 1:500 for 30 min, washed and stained with streptavidin conjugated horseradish peroxidase (BD Biosciences, San Diego, Calif.) at 1:1,000 for 30 min. Immunohistochemical staining was detected using the SK-4100 DAB kit, as per manufacturer's instructions (Vector Lab).