Signalstain diaminobenzidine dab substrate

The paraffin-embedded kidney slides were treated step-by-step by dewaxed, rehydrated, and antigen retrieval. Then the slides were incubated with 3% hydrogen peroxide for 10 minutes at room temperature and were blocked with 10% goat serum for 1 hour at 37°C. The sections were stained with p65 (1 : 100), p-p65 (Ser 536) (1 : 50), IκBα (1 : 100), and p-IκBα (Ser 32) (1 : 50) primary antibody (Cell Signaling Technology, Beverly, MA, USA) at 4°C overnight followed by SignalStain Boost Detection Reagent (Cell Signaling Technology, Beverly, MA, USA) for 30 min at room temperature. The sections were then treated with SignalStain diaminobenzidine (DAB) substrate (Cell Signaling Technology, Beverly, MA, USA), followed by counterstaining with hematoxylin and mounting. The Image-Pro Plus 6.0 software (Media Cybernetics, CA, USA) was used to calculate integrated optical density (IOD) values.

The paraffin-embedded rat kidney slides were dewaxed, rehydrated, and immersed in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity. Then, the slides were blocked with 10% goat serum for 1 h at 37°C and were stained with primary antibodies against fibronectin (FN) (dilution 1:200), type IV collagen (Col-IV) (dilution 1:200), and α-smooth muscle actin (α-SMA) (dilution 1:250) at 4°C overnight followed by SignalStain Boost Detection Reagent (Cell Signaling Technology, Beverly, MA, USA) for 30 min at room temperature. The sections were then treated with SignalStain diaminobenzidine (DAB) substrate (Cell Signaling Technology, Beverly, MA, USA) and counterstained with hematoxylin. The integrated optical density (IOD) values of the positive staining areas were measured by ImagePro Plus 6.0 software (Media Cybernetics, CA, USA). Five microscopic fields (200×) of each rat and three rats in each group were counted in a blinded manner.

Deparaffinized and hydrated sections (3 μm thick) were first pretreated with sodium citrate buffer (0.01 M, pH 6.0) in a water bath (95°C, 30 min) for unmasking of antigens, and then exposed to 3% hydrogen peroxide for 20 min to quench endogenous peroxidase activity. Thereafter, they were incubated in 10% goat serum for 30 min under room temperature, followed by overnight incubation with primary antibodies, including Bcl-2(1 : 200), p-p65 (Ser536) (1 : 50), and p65 (1 : 50) (Cell Signaling Technology, USA) at 4°C. Subsequently, the sections were washed and incubated with a SignalStain Boost IHC Detection Reagent (Cell Signaling Technology, USA) for 1 h, then, SignalStain diaminobenzidine (DAB) substrate (Cell Signaling Technology, USA) was added to develop a brown color. The sections were photographed under an optical microscope (Carl Zeiss, Germany). Three kidney specimens from each group were blindly captured across four equal microscopic fields (400x). For quantitative analysis, we performed integrated optical density (IOD) measurements on immunohistochemistry images to calculate average optical density (AOD) using the equation AOD = IOD/area. After defining the region of interest, the AOD of the selected area (IOD per unit area) was determined using Image-Pro Plus 6.0 software. The AOD represents the protein immunoreactivity within the renal tissue.