Rabbit anti α smooth muscle actin anti α sma

For immunohistochemical analysis, 5-μm sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked by the addition of 3% H₂O₂, in methanol. Antigen retrieval was performed using the heat induced epitope retrieval (HIER) method with a citrate buffer of 0.1 M, pH 6. Tissue sections were then incubated with rabbit anti-α-smooth muscle actin (anti-α-SMA, 1:500, Cell Signaling Technology, Danvers, MA, United States) overnight at 4°C. After two washes with PBS, slides were incubated with HiDef Detection amplifier Mouse and Rabbit reagent (Cell Marque, Rocklin, CA, United States) for 10 min, at room temperature. Sections were further washed with PBS and incubated with HiDef Detection HRP Polymer Detector solution (Cell Marque, Rocklin, CA, United States) for 10 min at room temperature. Finally, sections were washed twice with PBS, incubated with the DAB Chromogen kit (Cell Marque, Rocklin, CA, United States) for 5 min at room temperature, and counterstained with Hematoxylin. Dehydrated sections were mounted, and microphotographed on a light microscope, Nikon Eclipse E200. Quantification of α-SMA-positive (α-SMA⁺) areas was performed through use of Fiji software. Results were expressed as the mean intensity of α-SMA⁺ area per field.