Tissue was processed as previously described.3 (link),4 (link) Following removal of paraffin, followed by hydration, sections were stained with these antibodies: monoclonal-anti-MMP-9 (R&D Systems, Inc., Minneapolis, MN, USA; MAB911; 1:50 for 2 hours), monoclonal-anti-EGFR (Chemicon S/A, São Paulo, Brazil; CBL417; 1:40 for 60 minutes), and anti-S100A4 polyclonal antibody (Dako, Glostrup, Denmark; A5114; 1:2,500 for 90 minutes). In each case, a non-immune immunoglobulin (Ig)G1 negative control (Dako; X0931 clone DAK-GO1) was performed to eliminate false positive staining and a positive tissue control (surgically resected lung tissue) was also used. Bound antibodies were elaborated using peroxidase-labeled EnVision™ + (Dako; K4001) and liquid diaminobenzidine (DAB) + (Dako; K3468).
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Immunohistochemical Staining of Tissue Sections
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Corresponding Organization :
Other organizations : National Health and Medical Research Council, University of Tasmania, QIMR Berghofer Medical Research Institute, Newcastle University
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Variable analysis
independent variables
- Antibody type (monoclonal-anti-MMP-9, monoclonal-anti-EGFR, and anti-S100A4 polyclonal antibody)
- Antibody staining and binding
- Tissue processing as previously described
- Paraffin removal and hydration of sections
- Antibody incubation times (2 hours, 60 minutes, 90 minutes)
- Antibody concentrations (1:50, 1:40, 1:2,500)
- Positive tissue control (surgically resected lung tissue)
- Negative control (non-immune IgG1)
- Positive control: surgically resected lung tissue
- Negative control: non-immune IgG1 (Dako; X0931 clone DAK-GO1)
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