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 proteinA) BLV-positive fetal lamb kidney (FLK) cell lineBrown at left indicates positive diaminobenzidine endpoint immunoperoxidase reaction to detect digoxygenin incorporated into PCR product within FLK cellsFLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reactionOriginal magnification ×400B) BLV-negative cell line Tb₁Lu with (left) and without (right) primersNo reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific backgroundOriginal magnification ×400C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mixDark brown at left indicates positive cells, some surrounding lumens filled with milkLack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissueOriginal magnification ×100D) BLV-positive human tissue sample 010 reacted with tax primersDark brown at left indicates epithelial cells facing the lumen of a large cystLack of reactive cells in sample at right without primers indicates reaction was not a false positiveOriginal magnification ×100E) 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 ductOriginal magnification ×40F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assayBrown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stromaNote lack of reaction in sample at right with hybridoma medium substituted for primary antibodyOriginal magnification ×40All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).