Anti nf κb p65 antibody

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

Aβ_25-35 (Lot: Y-0044) was purchased from BIOSS Biotech Limited Company (Beijing, China). The Hoechst 33342/PI apoptotic assay kit was purchased from Sigma-Aldrich (St. Louis, MO, USA). The TUNEL apoptosis detection kit was purchased from BOSTER Biotech Limited Company (Wuhan, Hubei, China). The Annexin V-FITC/PI apoptotic detection kit was purchased from KeyGEN Biotech Corp., Ltd. (Nanjing, China). Rhodamine 123 was purchased from Yansheng biochemical reagent Co., Ltd. (Shanghai, China). The RT-PCR kit was purchased from TransGen Biotech Limited Company (Beijing, China). 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Sigma-Aldrich (Milan, Italy). Anti-NF-κBp65 antibody was purchased from Invitrogen (Carlsbad, CA, USA). The CO₂ incubator and ultra-low temperature freezer were purchased from SANYO Electric Co., Ltd. (Moriguchi, Japan). The laser scanning confocal microscope was purchased from Leica DMIRB (Rueil Malmaison, France). The flow cytometer was purchased from Becton-Dickinson Labware (Franklin Lakes, NJ, USA). The gel imaging and analysis system ChemiDocXRS were purchased from Bio-Rad Laboratories, Inc. (Berkeley, CA, USA). The TC-512PCR amplification instrument was purchased from TECHNE (London, UK).