E. coli P4 invades mammary epithelium in alveolar macrophage-depleted mice. Mammary glands of lactating C3H/HeN mice were infused with either PBS-loaded liposomes (A–B) or clodronate-loaded liposomes (D) followed by intramammary infection with E. coli P4. Fluorescence staining of paraffin embedded sections (A and D) or cryosections (B) with DAPI (blue) combined with Sytox orange (red) (A) or phalloidin-rhodamine (red) (B and D) of mammary tissues 48 h after bacterial challenge. GFP expressing bacteria (yellow arrows in B and D) are visible in the alveolar space of PBS-liposome treated mice (B) and inside epithelial cells and in the alveolar space in clodronate-liposome treated mice (D). Neutrophil recruitment into the alveolar space is clearly visible in both treatments (white arrows) 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 PBS-liposomes with clodronate-liposomes infused mice using Student’s t-test. Scale bars 50 μm (A–B), 15 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.)