Figure 4: The INT/BCIP chromogenic in situ hybridization method is more sensitive and shows less background staining than the TSA Plus Cyanine 5 Kit. (A) Detection of UNC5 mRNA in lamprey brainstem by fluorescent in situ hybridization (Cyanine5 Tyramide Signal Amplification Kit). Several identified reticulospinal neurons are labeled (white arrows), but background staining is high. (B) Detection of UNC5 mRNA by chromogenic in situ hybridization using the AP-INT/BCIP method. This method reveals more stained neurons, and background staining is much lower. (C) Enlarged pictures of the images in box in panel A. Some neurons (e.g., Mauthner and B3) are easily distinguished from background staining, but others are more difficult to differentiate with the TSA fluorescent in situ hybridization method (white arrowhead). Mth, Mauthner. (D) High magnification of labeled neurons in box in panel B. More bulbar reticulospinal neurons (e.g., B1, B2, B3, B4) are positively labeled with the chromogenic AP-INT/ BCIP method than with TSA fluorescent in situ hybridization. Note that non-specific background staining is much lower with chromogenic in situ hybridization.