Journal: Scientific Reports

Article Title: Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs

doi: 10.1038/s41598-020-61573-3

Figure Lengend Snippet: Colocalization between SINV RNA FISH and Broccoli. The images (objective lens 20×) show infected BHK cells (MOI = 5), fixed with 3.7% formaldehyde in PBS 6 h after infection. After adding DFHBI-1T, RNA FISH E1 + E2 (red) colocalizes well with Broccoli-DFHBI-1T (green) signal for both TEUTR4Br and TEds10Br. The scatter plot (X – red, Y – green) shows strong colocalization (R colocalization = 0.9049). Scale bar = 100 μm.

Article Snippet: Cells in each well were then incubated with two custom Stellaris RNA FISH probe sets each containing 48 short Quasar 570-labeled non-overlapping oligonucleotides against SINV E1 and E2 genes (LGC Biosearch Technologies), 25 nM each, in hybridization buffer (working buffer plus 10% dextran) at 37 °C overnight.

Techniques: Infection

Journal: Scientific Reports

Article Title: Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs

doi: 10.1038/s41598-020-61573-3

Figure Lengend Snippet: Rare discrepancy between RNA FISH and Broccoli signals. The images (objective lens 63×) show TEds10Br-infected BHK cells (MOI = 5), fixed with 3.7% formaldehyde in PBS at 6 h after infection. Although rare, some cells showed a discrepancy between FISH and Broccoli signals (white arrow), resulting in a separated cluster in the scatter plot (X – red, Y – green). Scale bar = 20 μm.

Article Snippet: Cells in each well were then incubated with two custom Stellaris RNA FISH probe sets each containing 48 short Quasar 570-labeled non-overlapping oligonucleotides against SINV E1 and E2 genes (LGC Biosearch Technologies), 25 nM each, in hybridization buffer (working buffer plus 10% dextran) at 37 °C overnight.

Techniques: Infection

Journal: Scientific Reports

Article Title: Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs

doi: 10.1038/s41598-020-61573-3

Figure Lengend Snippet: Live cell imaging of infected cells in mouse brain slices incubated with DFHBI-1T. ( A ) Channel mode and ( B ) lambda mode imaging of fresh TEds10Br-infected brain or ( C ) TE-infected brain. Broccoli-DFHBI-1T signal (green) in cells of the corpus callosum. ( D ) RNA E1 FISH imaging (red) of infected neurons in formalin-fixed brain tissue counterstained with DAPI. ( E ) Image of the 250 μm slice (McIlwain tissue chopper) of TEds10Br-infected mouse brain (3 days after infection) in a 35-mm glass-bottom dish in imaging medium containing DFHBI-1T with area of imaging for A, B indicated in the red square. Scale bar = 50 μm.

Article Snippet: Cells in each well were then incubated with two custom Stellaris RNA FISH probe sets each containing 48 short Quasar 570-labeled non-overlapping oligonucleotides against SINV E1 and E2 genes (LGC Biosearch Technologies), 25 nM each, in hybridization buffer (working buffer plus 10% dextran) at 37 °C overnight.

Techniques: Live Cell Imaging, Infection, Incubation, Imaging