Horseradish peroxidase conjugated secondary antibodies

Aorta samples were washed with phosphate-buffered saline (PBS, pH 7.4) prior to preparation of two cross sections during dissection. One cross-section was positioned with the luminal side facing downwards on a cup (Cryomold; Sakura Finetek, Torrance, California, United States) and covered with the Tissue-Tek optimal cutting temperature compound (O.C.T. Compound; Sakura Finetek). The aortic sample was placed on dry ice for at least 5 min, and frozen samples were stored at − 80 °C until sectioning was performed. The remaining cross-sections were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin prior to GRK2 immunohistochemical analysis. This was accomplished by sectioning tissue samples into 4-µm thick slices that were made to adhere to glass slides. GRK2 was detected in these sections using an anti-GRK2 rabbit monoclonal antibody (1:50, ST05-60; Novus, Littleton, Colorado, United States). Staining was visualized with the aid of horseradish peroxidase-conjugated secondary antibodies and 3,3’-diaminobenzidine (DAB; Nichirei Biosciences, Tokyo, Japan). Slides were counterstained with Harris modified hematoxylin, and subsequently dehydrated using ethanol solutions of incremental concentrations and xylene before being mounted under coverslips using PARA mount-N (Meiji Seika Pharma, Tokyo, Japan).