3 3 diaminobenzidine dab chromogen

Immuno-histochemical assays were used to determine CD163 and iNOS protein expression in the bladder tissue of rats. Tissues were obtained from rats with/without HPP treatment, fixed in 10% formaldehyde for 24 h, and then embedded in paraffin. The specimens were cut into 5-μm sections and roasted at 60°C for 2 h. Antigenic retrieval was performed in citric acid buffer (pH = 6.0) followed by washing with PBS and treatment with 3% H₂O₂ and 5% bovine serum albumin. Afterward, the sections were incubated with primary antibodies against CD163 and iNOS (dilutions of 1:50; Cell Signaling Technology) at 4°C overnight. Subsequently, they were incubated with secondary antibody for 30 min. Detection was performed using 3,3′-diaminobenzidine (DAB) chromogen (Maixin Biotech. Co. Ltd, Fuzhou, China), and the sections were counterstained with hematoxylin, dehydrated through graded alcohols, and cleared in xylene. Finally, pictures were taken under 200× magnification under a microscope (Ti2-E; Nikon). Immunostaining was evaluated by Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc., Silver Spring, MD, USA).

Immunohistochemical staining for the detection of polarization of macrophages was performed on Ventana’s Bench Mark XT automatic immunohistochemical staining machine using the avidin-biotin immunoperoxidase method. Briefly, the paraffin-embedded colon tissue sections were subjected to antigen retrieval and then were blocked and labeled overnight at 4°C with iNOS or CD206 antibody (1:200). After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Maixin, Fuzhou, China). The reaction was visualized using a 3,3′-diaminobenzidine (DAB) chromogen (Maixin).