Single molecule RNA fluorescence in situ hybridization (FISH) showing fixed brain sections on four separate slides demonstrating the lack of probe localization in non-immune tissue. We probed simultaneously for both TcRα and AID (a-c), individually for AID (probes labeled with Quasar 670 and imaged with Cy5 filter; pseudo colored red) (d-f), individually for TcRα (probes labeled with CalFluor Red 610 and imaged with Cy3 filter; pseudo colored green) (g-i), or for neither probe (negative control) (j-l). We show probe staining in shark thymus tissue as a positive control [m-o]. We counterstained all slides with DAPI (blue). We imaged fluorescence detected using each filter (DAPI, Cy3/Cy5) regardless of the probe used to illustrate that background fluorescence is not dependent on probe application. We obtained images of each fluorophore using 10x magnification and merged Z-stacked images together (DAPI fluorescence [a,d,g,j,m]; Cy3 (green) and Cy5 (red) fluorescence [b,e,h,k,n]; merged [c,f,I,l,o]). Scale bar 150 μm.