Immunohistochemical Detection of Amyloid Proteins

AApoAII and AA fibrils were also detected by immunohistochemistry (IHC) with specific rabbit antiserum against mouse ApoA-II or mouse AA, which were produced against guanidine hydrochloride-denatured AApoAII or AA fibrils in our laboratory [20 (link), 21 (link), 33 (link), 38 (link)]. Four-micron (4 μm) thick sections of fixed organs were treated with 3% H₂O₂ in methanol for 30 minutes (min) to inactivate endogenous peroxidase and were blocked with 5% bovine serum albumin in PBS. The sections were incubated overnight at 4°C with rabbit antisera against mouse ApoA-II (1 : 3000) and AA (1 : 3000) prepared in our laboratory [39 (link)] or 4-hydroxynonenal (4-HNE) (1 : 300, Abcam plc, Cambridge, UK), followed by incubation with the biotinylated secondary antibody (Abcam plc). Target proteins were identified by the horseradish peroxidase-labeled streptavidin-biotin method (DAKO, Glostrup, Denmark). In a negative control section, the first antibody was omitted to confirm the specificity of staining. To analyze the positive area in each organ quantitatively, the ratios of the positively stained area to a whole organ in randomly captured 5 areas under ×200 or ×400 magnification were measured using image processing program (NIH ImageJ software, version 1.61) [33 (link)].