Immunohistochemical Analysis of 4-HNE

Paraffin-embedded tissue sections were deparaffinized and rehydrated with graded ethanol dilutions. Antigen retrieval was applied to the tissue sections in a pressure cooker using an Antigen Retrieval Machine (2100 Retriever) with EDTA Buffer pH 9. Immunohistochemistry was performed using the Dako REAL Detection Kit System, Alkaline Phosphatase/RED, Rabbit/Mouse Kit (Dako Cat.# S2022, distributed by Agilent Technologies AG) according to the manufacturer’s instructions and as previously described [78 (link)]. For washing steps, the Dako washing buffer diluted with 1:10 with distilled water was used, and the Dako dual endogenous enzyme block (S2003) was applied to the tissue section before staining to inhibit endogenous phosphatase activity. Tissue sections were incubated with mouse monoclonal anti-human 4-HNE (1:50 dilution; clone 12F7, Invitrogen cat. #MA5-27570, distributed by ThermoFisher Scientific) in Dako antibody diluent S2022, followed by a biotin-streptavidin system coupled with alkaline phosphatase and counterstained with Hematoxylin dye (Dako Cat. #S2020) and mounted with Aquatex (Merck Cat. #1.08562.005, distributed by Sigma-Aldrich, Buchs, Switzerland). The images were visualized using a light microscope (Carl Zeiss Micro Imaging, Jena, Germany).