Figure 7

Effect of detergent in washing buffer on staining of prolonged formalin-fixed human ductal breast cancer tissue with the #394 monoclonal uPA antibody and a control tri-nitro-phenyl (TNP) antibody. Sections from paraffin-embedded specimens, fixed for 24 hours at room temperature, were incubated with the #394 monoclonal antibody against uPA (10 μg/ml, Panels a, b) or the negative control monoclonal antibody against TNP (10 μg/ml, Panels c, d), and these were immunohistochemically processed without (Panels a, c) or with (Panels b, d) detergent (0.5% Triton X-100) in the washing buffer. No staining is seen with #394 and anti-TNP using detergent containing washing buffer (Panels b, d), whereas staining of cancer cells is observed with both #394 and anti-TNP when detergent is omitted from the washing buffer (Panels a, c). Note that, with the prolonged fixation used in this experiment, immunostaining for uPA is not found in the stromal cells of detergent-washed sections. This lack of specific uPA staining of prolonged formalin-fixed tissue is in agreement with results obtained with other uPA antibody preparations (Figure 5 and Table 1). In contrast, the standard procedure, including fixation for 1 hour at 4° C and detergent wash, results in staining that is confined to stromal cells when 40 μg/ml of the #394 antibody is used (Figure 2B, Panel d). As discussed in the text, we consider the staining of the cancer cells shown in Panel a to be nonspecific. Bar: 50 μm.