Diaminobenzidine dab chromogen

Five μm sections from each paraffin-embedded tissue block were prepared from cervical cancer and inflammatory lesions. Immunohistochemical staining was performed on paraffin-embedded tissue sections as described by Rahmani et al., 2015 [14 (link)]. In brief, deparaffinization of all sections was made through a series of xylene solutions, and rehydration was performed through graded ethanols. Also, endogenous peroxidase activity was blocked by pre-treatment of sections with 0.3% hydrogen peroxide for 20 minutes. Antigen retrieval was performed using a sodium-citrate buffer (pH 6.0), in a microwave oven for 30 minutes. Additionally, protein blocking agent (Abcam, USA) was applied for 10 minutes to reduce nonspecific binding. VEGF and Her-2 antihuman monoclonal antibodies (Abcam, Cambridge, MA, USA) were used as primary antibodies followed by the secondary biotinylated antibody (Abcam, USA). Detection of immunostaining for VEGF and Her-2 protein was performed using the streptavidin-biotin method. Diaminobenzidine (DAB) chromogen (Abcam, USA) was applied then sections were counterstained and mounted with DPX. Negative controls (omission of primary antibody) and positive controls were used to verify the quality of staining.