SIV ISH was performed on serial sections using next-generation ISH RNAscope, as previously described (32 (link)). The slides were scanned at high magnification (×400) using the Aperio AT2 digital Whole Slide Scanning System (Leica Biosystems), yielding high-resolution data from the entire tissue section. After regions of interest (ROIs) were determined and mapped on the digital Aperio scans of each tissue, laser capture microdissection was performed on the subjacent serial sections aligned to the specified ROIs using an ArcturusXT, Nikon TE2000 Microscope laser capture microdissection system. The targeted areas were captured on different caps (CapSure Macro LCM Caps, Applied Biosystems), as previously described (42 (link)). Each tube was inverted to allow total immersion of the tissue in the protease K solution and incubated at 65°C for 20 hours. The samples were then used for viral sequencing, as described above.
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Spatial mapping of viral RNA by Next-Gen ISH
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Corresponding Organization : Frederick National Laboratory for Cancer Research
Other organizations : UNSW Sydney, University of Sydney, Center for Disease Dynamics, Economics & Policy, Pennsylvania State University
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Variable analysis
independent variables
- Laser capture microdissection of specified regions of interest (ROIs) on the tissue sections
- Viral sequencing of the captured tissue samples
- Serial sections used for SIV ISH analysis
- Scanning of the slides at high magnification (×400) using the Aperio AT2 digital Whole Slide Scanning System
- Alignment of the serial sections to the specified ROIs
- Capture of the targeted areas on different caps
- Incubation of the samples in protease K solution at 65°C for 20 hours
- No positive or negative controls were explicitly mentioned in the provided information.
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