Detection of Zika Virus in Tissues

Tissues were placed into 10% neutral buffered formalin for 3 days and then stored in 70% ethanol prior to processing. Sections were cut at 4 microns and stained with hematoxylin and eosin or by in situ hybridization (ISH) for ZIKV RNA. The ISH assay was performed using the ViewRNA ISH tissue assay (ThermoFisher) with a probe set targeting the ZIKV (+) strand RNA genome. As a negative control, tissue sections from an uninfected female mouse were stained using the ZIKV probe set. As a positive control, tissues were stained using probes against the housekeeping murine mRNAs GAPDH, PIPB, and β-actin. Tissue sections were de-paraffinized and underwent pre-treatment and protease treatment for ten minutes each. Positive staining (red punctate staining) was detected using an alkaline-phosphatase label probe. The slides were counterstained with Gill’s Hematoxylin I. The IHC assay was performed using a polymer-based indirect immunoalkaline phosphatase detection system with colorimetric detection of antibody/polymer complex with Fast Red chromogen. The primary antibody was a rabbit polyclonal antibody generated against ZIKV VLPs^{21 (link)}. Negative controls for IHC comprised both uninfected tissues incubated with the primary antibody, and infected tissues incubated with normal rabbit serum in place of the primary antibody.