Secondary antibody conjugated to horseradish peroxidase hrp

Paraformaldehyde-fixed, paraffin-embedded (PPFE) tissue sections were deparaffinized, rehydrated, and then incubated in a microwave oven with 0.01 M citrate buffer, pH 6.0 for 10 min for antigen retrieval. Endogenous peroxidases were blocked with 3% H₂O₂ for 15 min. Nonspecific epitopes were blocked with normal horse serum (Jackson ImmunoResearch, West Grove, PA) for 1 h. The sections were incubated overnight at 4°C with antibodies against one of the following proteins: LOX, MMP9, MMP2, LYVE1, VEGF, and S100A8 (all from Abcam, Cambridge, MA), S100A9, IL-33 (R&D Systems, Minneapolis, MN), cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA), Ki67 (OriGene Technologies, Rockville, MD). This was followed by incubation with secondary antibody conjugated to horseradish peroxidase (HRP) (Bio-Rad, Hercules, CA) for 1 h at room temperature. Antigen signals were detected using the 2-Solution Diaminobenzidine (DAB) Kit (Invitrogen, Frederick, MD), counterstained with hematoxylin, mounted in Acrymount (StatLab, Mckinney, TX), and visualized under a light microscope.