RNAScope® assay and RNA FISH were performed as previously described16 (link). RNAScope® robes targeting LINK-A (Cat# 412027), BCAR4 (Cat# 407777) or HOTAIR (Cat# 312347) were custom designed or purchased from Advanced Cell Diagnostics. LNA™ FISH probes targeting LINK-A and control probe targeting Beta-Actin (300512-04) were purchased from Exiqon (sequences were listed in Oligonucleotide sequences, probes and primers section).
For immuno-RNA FISH, the slide from RNA FISH was further blocked with blocking buffer [1 × PBS, 5% BSA, 0.3% Triton X-100] for 1 hour at room temperature followed by incubation with primary antibodies (diluted 1:200) for 1 hour at room temperature. After incubation with fluorochrome-conjugated secondary antibodies for 1 hour at room temperature in dark, the slide was washed and mounted for detection. Immunofluorescence and immunohistochemistry were performed as previously described16 (link).
The quantification of RNAScope® staining densities was measured by RNAscope® SpotStudio v1.0 Software (Advanced Cell Diagnostics). The quantification of IHC staining density was performed by Image-Pro plus 6.0 (Media Cybernetics) and calculated based on the average staining intensity and the percentage of positively stained cells.
For immuno-RNA FISH, the slide from RNA FISH was further blocked with blocking buffer [1 × PBS, 5% BSA, 0.3% Triton X-100] for 1 hour at room temperature followed by incubation with primary antibodies (diluted 1:200) for 1 hour at room temperature. After incubation with fluorochrome-conjugated secondary antibodies for 1 hour at room temperature in dark, the slide was washed and mounted for detection. Immunofluorescence and immunohistochemistry were performed as previously described16 (link).
The quantification of RNAScope® staining densities was measured by RNAscope® SpotStudio v1.0 Software (Advanced Cell Diagnostics). The quantification of IHC staining density was performed by Image-Pro plus 6.0 (Media Cybernetics) and calculated based on the average staining intensity and the percentage of positively stained cells.