Diaminobenzidine dab chromogen system

Ovarian healthy (n = 12) and cancer (n = 8) tissues were rapidly washed with 0.1 M phosphate buffered saline (PBS) at pH 7.4 and fixed for 4–12 hours in 4% buffered formalin at 4°C. The specimens were then dehydrated in ethanol and embedded in paraffin wax. Serial sections of tissue were deparaffinized and rehydrated through graded ethanol. Antigen retrieval was performed by microwave pretreatment in 10 mmol/L citrate buffer (pH 6.0) for 5 minutes four times, followed by cooling in a cold water bath. Nonspecific binding was blocked with 3% (v/v) bovine serum albumin (BSA) in PBS for 30 minutes at room temperature. The sections were incubated with anti-human tenascin-X (diluted in 3% BSA-PBS, H-90, Santa Cruz Biotechnology, Labforce, Nunningen, Switzerland, or AF6999 from R&D) or with control IgG (sc-2027, Santa Cruz Biotechnology, Labforce, Nunningen, Switzerland) overnight at 4°C. Sections were then washed with PBS and incubated with goat anti-rabbit or rabbit anti-sheep IgG-HRP (dilution 1/500) for 1 hour. After washing, sections were stained with diaminobenzidine (DAB) chromogen system (Dako, Baar, Switzerland). The stained tissue was scored independently by 2 experts. The intensity of staining was scored as absent (0), weak (1), moderate (2), and intense (3).

Ovarian cancer tissues were rapidly washed with 0.1 M phosphate buffered saline (PBS) at pH 7.4 and fixed for 4–12 hours in 4% buffered formalin at 4°C. The specimens were then dehydrated in ethanol and embedded in paraffin wax.
5 sections of human healthy ovarian tissue and 5 sections of human ovarian cancer tissue were deparaffinised and rehydrated through graded ethanol. Antigen retrieval was performed by microwave pre-treatment in 10 mmol/l citrate buffer (pH 6.0) for 5 minutes four times, followed by cooling in a cold water bath. Non-specific binding was blocked with 3% (v/v) bovine serum albumin (BSA) in PBS for 30 minutes at room temperature. The sections were incubated with polyclonal rabbit anti-human PAR-4 (R-334) or with control IgG (dilution 1/500 in 3% BSA-PBS) overnight at 4°C. Sections were then washed with PBS and incubated with goat anti-rabbit IgG-HRP (dilution 1/500) for 1 hour. After washing, sections were stained with diaminobenzidine (DAB) chromogen system (Dako, Baar, Switzerland). The stained tissue was scored independently by 2 experts. The intensity of cytoplasm and nucleus staining was scored as absent (0), weak (1), moderate (2) and intense (3).

We evaluated the levels of 3-NTY as a marker of kidney nitrosylated oxidation products caused by formation of peroxynitrite. Five micrometer sections of the kidney were initially quenched of endogenous peroxidase and incubated with 1:200 rabbit polyclonal anti-3-NTY antibody overnight (Chemicon, Temecula, CA) [40 (link), 41 (link)]. Sections were washed and incubated with appropriate secondary antibody and signals visualized by diaminobenzidine (DAB) chromogen system (DAKO, Carpinteria, CA). Using a 50i Nikon microscope, five randomly selected 10× bright-field images from each section were captured with a CoolSNAP cf camera. Signal intensities of brownish color, which is indicative of the 3-NTY level, were quantified by MetaVue software.