Lethal Nipah Virus Infection Induces Rapid Overexpression of CXCL10

Mathieu, Cyrille; Guillaume, Vanessa; Sabine, Amélie; Ong, Kien Chai; Wong, Kum Thong; Legras-Lachuer, Catherine; Horvat, Branka

Autopsy brain tissue was obtained from 3 fatal acute Nipah cases from the outbreak in Malaysia in 1999, fixed in 10% buffered formalin and paraffin-embedded and from two control brain tissues from patients that succumbed to non-neurological diseases. Sections (4 mm) were deparaffinized with xylene and rehydrated through graded alcohol and distilled water. Antigen retrieval was performed in 10 mM trisodium citrate buffer, pH = 6, using waterbath (99uC, 20 min). Slides were then washed 3 times in PBS 16. Endogenous peroxidase was blocked using H2O2, 3% in PBS, for 20 min at Room temperature (RT). Slides were then washed 2 times in PBS 16 and additional blocking was performed using goat serum (1:20 in PBS) for 20 min at RT. Primary rabbit anti-human CXCL10 (Peprotech), was applied diluted at 4 mg/ml, overnight at 4uC. After 3 washes in PBS, slides were incubated with secondary goat anti-rabbit conjugated with HRP (Promega) 1:500 for 1-2 hours at RT. Sections were then developed with AEC kit (VECTOR) for 25 min, washed in distilled water and counterstained with Harris hematoxylin solution (Sigma-Aldrich) 1:3 in PBS, 30 s. After one wash in water (3 min) sections were mounted with DakoCytomation faramount aqueous mounting medium and coverslipped. Slides were analysed using Axioscope microscope equipped with Zeiss Axiovision software (Zeiss Geramny).