Immunohistochemical Analysis of NF-κB and TLR4

Cardiac sections were used for IHC staining. Hence, 3% hydrogen peroxide (H₂O₂) in methanol was used to block the endogenous peroxidase enzyme in the obtained sections at 21–25 °C for 30 min, followed by rinsing three times in phosphate-buffered saline (PBS). Afterward, the sections were incubated with the antibodies, stored overnight at 4 °C in a humidified chamber, and then goat anti-rabbit-horseradish peroxidase (HRP)-conjugated IgG antibody (1:1000; cat. no. ab6721; Abcam) was added for 1 h at 37 °C. The primary antibodies against rabbit polyclonal anti NF-κBp65 antibody (Thermo Fisher Scientific, USA), and anti-TLR4 antibody (Abcam, UK), were used following the procedures described previously [33 (link)]. Finally, the sections were developed with 1% diaminobenzidine for 5 min, counterstained with 1% hematoxylin for 2 min at 21–25 °C, and mounted with neutral gum. Cardiac slices were imaged using a microscope fitted with a digital camera (Nikon Instruments Inc., Tokyo, Japan). NIS-Elements software was used for the semi-quantitative analysis of NFκB and TLR4. First, the area of the immunohistochemical reaction in the picture was selected. Then, the average optical density in the selected area of each picture was measured. Positive cells were counted under 400× magnification observing 10 consecutive non-overlapping fields per animal in a blinded manner