Figure 3.

Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint immunoperoxidase reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb₁Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).