SIV RNA Detection in Tissues

All tissues in O.C.T were cut at 10 µm on cryostat (LEICA CM 3050 S) and put on slides (Thermo Scientific). These slides were conserved at −20 °C. The epitope revelation protocol was realized by two methods. For detection of SIVmac RNA, we followed the RNAscope protocol with SIVmac251-gag probe utilization (Advanced Cell Diagnostics Europe) and ACD HybEZ Hybridization system (Advanced Cell Diagnostics Europe, 310013). For detection of SIVagm RNA, the SIVagm probe was made based on the SIVagm.sab92018 backbone, as described in a previous study^{17 (link)}. The primary antibodies (Supplementary Data 4; 1:200) were added overnight at 4 °C. Then we washed the slide during 1 h in PBS and added secondary antibodies (Supplementary Data 4; 1:200) and DAPI (Supplementary Data 4; 1:1000) for 1 h at 4 °C. We washed the slide during 1 h in PBS and we added mounting medium (Invitrogen, 00-4958-02). We finally dropped off the cover (Fisher Scientific, Dutscher) from the slide with mounting medium. The fluorescence staining was observed and captured by a spinning-disk on three tissue sections per monkey (Yokagawa, CellVoyager CV1000). We analyzed these images by ImageJ (Fiji).