Immunohistochemistry Problem

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bndelong
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Immunohistochemistry Problem

I have been working on a proliferation study on developing lungs of rodents and am having a problem getting specific staining of proliferating lung epithelial cells. The animals are injected with BrdU 2hrs prior to harvesting the lungs. They are fixed in 1%PFM and embedded in paraffin and sectioned. I am getting labelled epithelial cells but I am also getting nonspecific labeling of something in the parenchyma. It initially appears to be some sort of peroxidase but the negative controls (+ primary antibody/ - secondary antibody or vice-a-versa) are clean. Moreover, the labeling in the parenchyma is non-nuclear. The only idea I have is that there is some amount of free BrdU in the surrounding tissue which is being picked up by the antibodies. Any ideas?