Rnascope red 2.5 kit

In situ hybridization (ISH) was conducted with tissues fixed in 4% PFA, and alcohol processed and paraffin embedded. ISH probes against Zika genome were purchased commercially (Advanced Cell Diagnostics, Cat No. 468361, Newark, California, USA). ISH was performed using the RNAscope^® Red 2.5 Kit (Advanced Cell Diagnostics, Cat No. 322350) according to the manufacturer’s instructions. Briefly, after deparaffinization with xylene, a series of ethanol washes, and peroxidase blocking, sections were heated in boiling antigen retrieval buffer for 15 minutes and then digested by proteinase K (2.5 μg/ml, to completely cover the section) for 16 minutes at 40°C. Sections were exposed to ISH target probe and incubated at 40°C in a hybridization oven for 2 h. After rinsing, ISH signal was amplified using company-provided Pre-amplifier and Amplifier conjugated to horseradish peroxidase (HRP), and incubated with a red substrate-chromogen solution for 10 min at room temperature.

In situ hybridization was conducted on cross sections of placental cotyledons as described previously (22 (link)). Briefly, tissues were fixed in 4% PFA, alcohol processed, and paraffin embedded. Commercial ISH probes against the ZIKV genome (catalog no. 468361; Advanced Cell Diagnostics, Newark, CA, USA) were used. ISH was performed using the RNAscope Red 2.5 kit (catalog no. 322350; Advanced Cell Diagnostics) according to the manufacturer's instructions.

ISH probes against the ZIKV genome were commercially purchased (cat# 468361, Advanced Cell Diagnostics, Newark, CA). ISH was performed using the RNAscope Red 2.5 kit (cat# 322350, Advanced Cell Diagnostics, Newark, CA) according to the manufacturer’s protocol. After deparaffinization with xylene, a series of ethanol washes, and peroxidase blocking, sections were heated with the antigen retrieval buffer and then digested by proteinase. Sections were then exposed to the ISH target probe and incubated at 40°C in a hybridization oven for two-hours. After rinsing, the ISH signal was amplified using the provided pre-amplifier followed by the amplifier-containing labeled probe binding sites, and developed with a Fast Red chromogenic substrate for 10 minutes at room temperature. Sections were then stained with hematoxylin, air-dried, and mounted.

ISH probes against the ZIKV genome were commercially purchased (cat# 468361, Advanced Cell Diagnostics, Newark, CA). ISH was performed using the RNAscope^® Red 2.5 kit (cat# 322350, Advanced Cell Diagnostics, Newark, CA) according to the manufacturer’s protocol. After deparaffinization with xylene, a series of ethanol washes, and peroxidase blocking, sections were heated with the antigen retrieval buffer and then digested by proteinase. Sections were then exposed to the ISH target probe and incubated at 40°C in a hybridization oven for two-hours. After rinsing, ISH signal was amplified using the provided pre-amplifier followed by the amplifier conjugated to alkaline phosphatase l, and developed with a Fast Red chromogenic substrate for 10 minutes at room temperature. Sections were then stained with hematoxylin, air-dried, and mounted prior to imaging using a Leica DM 4000B microscope equipped with a Leica DFC310FC camera and Surveyor software (Version 9.0.2.5, Objective Imaging, Kansasville, WI).