E. coli P4 invades mammary epithelium of iNOS −/− mice 48 h after intramammary infection. Fluorescence staining of cryosections (A and D) or paraffin embedded section (B) with DAPI (blue) combined with phalloidin-rhodamine (red) (A and D) or Sytox orange (red) (B) of mammary tissues 48 h after bacterial challenge. Numerous bacteria are visible in the alveolar space of the wild type (wt) C57BL/6 mouse (yellow arrow in B) while GFP expressing bacteria are visible inside epithelial cells in iNOS −/− mice (yellow arrows in D). Similar neutrophil recruitment (white arrows) into the alveolar space is clearly visible in both C57BL/6 (B) and iNOS −/− (A and D) mice, which is also reflected by relative (ratio of challenge to control gland) MPO activity (C). Data are mean and S.E., n ≥ 6/group and p value compared iNOS −/− with wt mice using Student’s t-test. Scale bars 50 μm (A-B) and 20 μm (D). All images are representative of the entire sample and the histological morphology and pathology results were very similar for each gland in a given mouse and between mice. (For a color version of this figure please consult www.vetres.org.)