Subcellular localization of LncRNA-HIT and Gapdh transcripts in limb mesenchyme using single molecule RNA fluorescent in situ hybridization (FISH).

(A-D)LncRNA-HIT transcripts are detected in the nucleus of undifferentiated limb mesenchyme at E 11.0. Red signal = detection of LncRNA-HIT probe sets labeled with CalFluor610. Blue signal = DAPI staining of the nuclear DNA. (E and F) Gapdh transcripts are detected in the cytoplasm of undifferentiated limb mesenchyme. Green signal = detection of Gaph probe sets labeled with CalFluor610 and pseudo-colored green. Blue signal = DAPI staining of the nuclear DNA. (G) Negative control using RNase A prior to hybridization with the LncRNA-HIT probe sets reveals no detected LncRNA-HIT (red signal) in the nucleus confirming the detected signal in panels A-D represent hybridization with the LncRNA-HIT transcript. The nuclear DNA was unaffected by the RNase A treatment and stained positively with DAPI (blue signal). (H) Negative control using RNase A prior to hybridization with the Gapdh probe sets reveals no detected Gapdh transcript (green signal) in the cytoplasm confirming the detected signal in panels E and F represent hybridization with the Gapdh transcript. The nuclear DNA was unaffected by the RNase A treatment and stains positively with DAPI (blue signal). Bar = 10 μm.