3 3 diaminobenzidine dab chromogen substrate solution

Tissue samples were fixed overnight in 10% neutral buffered formalin, embedded in paraffin, and processed as 5 µm thick sections. Deparaffinized sections were briefly heated for 4 min in a pressure cooker containing 10 mM citrate buffer (pH 6.0) for antigen retrieval. Subsequent procedures were performed at room temperature. The sections were pretreated with 3% H 2 O 2 in 0.1 M Tris-buffered saline (TBS, pH 7.4) for 30 min to quench endogenous peroxidases, followed by treatment with Protein Block Solution (DAKO) for 20 min and incubation with anti-ERβ antibody for 30 min in a humidified chamber. After washing with 0.1 M TBST (0.1 M TBS containing 0.01% Tween 20), the sections were incubated with EnVision anti-rabbit (DAKO) polymer for 30 min. Peroxidase bound to the antibody complex was visualized by treatment with 3, 3'-diaminobenzidine (DAB) chromogen substrate solution (DAKO). The DAB reaction was monitored under a microscope to determine optimal incubation time and stopped with several washes of 0.1 M TBS. Immunolabeled sections were dehydrated in a graded ethanol series, defatted in xylene, and mounted, followed by examining with an Olympus BX51 microscope (Olympus, Japan) under bright-field illumination. Images were acquired with an Olympus DP 70 camera (Olympus).

Formalin-fixed paraffin-embedded sections were deparaffinized and boiled for 20 min in 10mM citrate buffer (pH 6.0). Sections were washed in TBST and exposed to 0.3% H 2 O 2 for 5 min to quench endogenous peroxidases. Immunohistochemistry was performed using the following antibody: rabbit anti-8OHdG polyclonal antibody (1:100, BIOSS, Woburn, MA, USA). Concentration-matched rabbit IgG was used as an isotype-negative control. The sections were blocked with Dako proteinblock (Dako, Carpinteria, CA, USA) for 10 min and incubated with primary antibody overnight. The slides were then incubated with horseradish peroxidase anti-rabbit Envision-system followed by a 3.3'-diaminobenzidine (DAB) substratechromogen solution (Dako) and counterstained with Harris hematoxylin. The slides were examined using a Leica photomicroscope linked to a DFC 480 digital camera (Leica, Wetzlar, Germany).
The quantitation was performed by capturing 6-10 non-overlapping fields of renal cortex from stained sections. Areas of brown staining reflecting 8-OHdG were highlighted using a selective color tool and the proportional area of the field with their respective color range was quantified using Image J.