Hidef amplifier

Four-micron histological sections were placed on ColorFrost Plus slides (Thermo Scientific, Waltham, MA, USA) at 58 °C for two hours. Antigenic recovery was performed under pressure (Cuisinart Pressure Cooker Model CPC-600) with Trilogy™ (Cell Marque, Rocklin, CA, USA) at 1:100 dilutions for 15 min at 125 °C. Endogenous peroxidase was blocked with 9% H₂O₂ diluted in methanol for 15 min. The sections were delineated with Dakopen (SDL, Des Plaines, IL, USA), and the tissues were covered with the antibody diluted 1:100 with anti-dengue 1 + 2+3 + 4 (ab26837, Abcam, Cambridge, UK) for one hour. HiDef Amplifier (Cell Marque) was added for 10 min at room temperature. HiDef HRP Polymer Detector (Cell Marque) was added for 10 min at room temperature. The tissue was covered with the Chromogen Liquid DAB + Substrate Chromogen System (Dako North America, Carpinteria, CA, USA) and stained with hematoxylin for one minute. As a negative control, the anti-dengue antibody was replaced with 1% phosphate-buffered saline (PBS). As positive controls, brain samples from suckling mice with an intracranial inoculation of DENV-2 were used. PCR-negative specimens of the same species were included.