Tissue blocks were cut for microscopy analysis. Following ex vivo challenge or not, explants were harvested at different time points and fixed in a 10% formalin solution (Sigma-Aldrich, St. Louis, MO, USA) for 1 to 3 days at room temperature prior to analysis. RNAscope and immunohistochemistry were performed as previously described [27 (link)]. Microscopy analysis was performed with a Leica Bond-Max system (Leica, Wetzlar, Germany) and image analysis with Pannoramic Viewer software (3DHistec, Budapest, Hungary).
For RNAscope ISH, fixed tissue blocks were embedded in paraffin wax (VWR) using previously reported procedures [92 (link)]. Briefly, 4 µm thick sections were mounted on poly-L lysine-coated slides (VWR) and, prior to treatment, de-waxed in xylene and re-hydrated via graded ethanol:water solutions. In situ ZIKV RNA detection was performed using the RNAscope 2.5 HD manual DAB detection system (Advanced Cell Diagnostics, Newark, CA, USA) and a combination of two ZIKV-specific probes (463781 and 464531) in accordance with the manufacturer’s instructions. Negative (DaPB 310043) and positive (Hs-PPIB 313901) control probes were used to assess technique efficiency. Sections were stained with hematoxylin for the detection of cellular nuclei.
For RNAscope ISH, fixed tissue blocks were embedded in paraffin wax (VWR) using previously reported procedures [92 (link)]. Briefly, 4 µm thick sections were mounted on poly-L lysine-coated slides (VWR) and, prior to treatment, de-waxed in xylene and re-hydrated via graded ethanol:water solutions. In situ ZIKV RNA detection was performed using the RNAscope 2.5 HD manual DAB detection system (Advanced Cell Diagnostics, Newark, CA, USA) and a combination of two ZIKV-specific probes (463781 and 464531) in accordance with the manufacturer’s instructions. Negative (DaPB 310043) and positive (Hs-PPIB 313901) control probes were used to assess technique efficiency. Sections were stained with hematoxylin for the detection of cellular nuclei.
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