Rnascope 2.5 hd manual dab detection system

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.

In situ RNA detection was performed using the RNAscope 2.5 HD manual DAB detection system (Advanced Cell Diagnostics, 322300) and a SARS-CoV-2 specific probe (V-nCoV2019-S-sense 845701/V-nCoV2019-S 848561) in accordance with manufacturer’s instructions. Negative (DaPB 310043) and positive (Hs-UBC 310041/Mfa-UBC 461331) control probes were used to assess technique efficiency. Equivalent tissue sections from infection naïve cynomolgus macaques and hamsters were stained with SARS-CoV-2 specific, positive or negative control probes to determine species tissue cross-reactivities. Sections were manually counter stained with haematoxylin.

Samples covering a range of tissue compartments including main organs, lymphoid tissues, reproductive system, peripheral and central nervous system, including the brain and skin were collected at post-mortem, fixed in 10% formal saline (Sigma) and embedded in paraffin wax (VWR) using previously reported procedures^{51 (link)}. Four micron 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, 322300) and a combination of two ZIKV-specific probes (463781 and 464531) in accordance with manufacturer’s instructions. Negative (DaPB 310043) and positive (Hs-PPIB 313901) control probes were used to assess technique efficiency. Equivalent tissue sections from infection naïve RM and tamarins were stained with ZIKV-specific, positive or negative control probes to determine NHP tissue cross-reactivities. Sections were manually counter stained with haematoxylin. RNAscope positive cells were quantified by counting all positive cells within each tissue section and conversion to mean number of positive cells/mm². Grading definitions were generated using 10 random fields of view (x10 lens and x10 eye-piece magnification; equivalent to an area of 2.2 mm²).