Envisiontm detection system

Five-micrometer heart sections were prepared and mounted on aminopropyltriethoxysilane (APES) coated slides. After dewaxing with xylene and rehydrating with graded alcohol, slides were placed in 0.01 M citrate buffer solution (pH = 6.0) and pre-treatment procedures to unmask the antigens was performed in a water bath at 95°C for 30 min. Then, sections were treated with peroxidase block for 60 min followed by protein block for 60 min. Sections were incubated with anti-Nrf2 (rabbit polyclonal, 1:100, Abcam, United States) for 1 hour at room temperature. After conjugation with primary antibodies, sections were incubated with secondary antibody (EnVisionTM Detection System, DAKO, Agilent, United States) for 20 min at room temperature followed by addition of DAB chromogen (EnVisionTM Detection System, DAKO, Agilent, United States) and counter staining done with hematoxylin. Appropriate positive controls were used. For negative control, the primary antibody was not added to sections. Positive and negative controls were used in every batch of slides that were stained (not shown in figures) (Nemmar et al., 2022 (link)).

β-catenin expression in tissues without polyps was analyzed using IHC. A piece of tissue from the internal end of distal colon, approximately 1 cm wide, was collected from rats in groups M7 to M10. Half of each tissue sample was fixed in 4% neutral buffered formalin for 24 h, paraffin embedded and cut into 4 μm thick sections. IHC of colon tissues was performed by using an EnVision^TM Detection System (Dako, Troy, MI, USA), according to manufacturer’s instructions. Briefly, tissue sections were mounted onto silanized charged slides and allowed to dry for 1 h at room temperature, followed by 1 h in a 60 °C incubator. After deparaffinization and rehydration, slides were incubated with a proteinase K solution (20 μg/mL⁻¹) for 5 min. After washing with distilled water, tissue sections were immersed in 3% H₂O₂ for 5 min to block endogenous peroxidase, followed by additional washing with buffer. Slides were incubated with a rabbit anti-β-catenin primary antibody (Abcam, Cambridge, UK) for 30 min in a humid chamber. After 2 rinses in buffer, slides were incubated with the detection system for 30 min. Staining was visualized with a 2,4-diaminobutyric Acid (DAB) substrate chromogen solution. Sections were dehydrated, mounted with cover glass, and observed using light microscopy (DFC425C, Leica, Wetzlar, Germany).

Immunostaining was applied on 5 μm thick paraffin sections. After deparaffinization and rehydration, the sections were treated by microwave for 20 min at 400 W in citrate buffer (pH 6.0). After antigen retrieval, samples were incubated for 1 hour at room temperature with primary antibody for nestin (Santa Cruz, USA, sc-23927, dilution 1:100). Sections were then treated with EnVisionTM Detection System (DAKO, Germany) using 3-amino-9-ethylcarbazole (AEC) as substrate, and counterstained with hematoxylin. Negative controls were performed by omitting the first antibody and stained by the EnVisionTM method, and for mouse monoclonal antibodies as isotype control mouse IgG1 (ab91353, Abcam, UK) antibody was also used. The slides were evaluated using a light microscope BX53 with DP12-CCD camera (Olympus, Germany).