Table 1.

Application of different detection systems in two-color WISH

Detection System 1	Detection System 2	Applications
POD-TSA- DyLight633	POD-TSA-FAM	▪ Routinely used for fluorescent co-localization analysis of two mRNAs at cellular resolution ▪ If one of the two probes cannot be detected by POD-TSA, combine POD and AP detection
POD-TSA-FAM	AP-Fast Red	▪ If one probe cannot be visualized by POD-TSA this probe may be detected by AP-Fast Red allowing for long staining times with high signal-to-noise-ratios ▪ Combine fluorescence and DIC optics, if histological context is required ▪ Fast Red is preferably used for the more locally distributed mRNA
POD-TSA-FAM	AP-Fast Blue	▪ If there is significant bleed-through between TSA-FAM and AP-Fast Red, AP-Fast Blue as second detection system may be applied instead ▪ If the weaker probe cannot be detected by TSA-FAM or Fast Red, prolonged Fast Blue staining may be helpful for visualization ▪ Combine fluorescence and DIC optics, if histological context is required ▪ Fast Blue is preferably used for the more locally distributed mRNA
AP-Fast Blue	AP-Fast Red	▪ For fluorescent visualization of non-overlapping expression domains ▪ For colorimetric detection of two mRNAs ▪ Permits long term storage of stained embryos
AP-Fast Red	AP-BCIP/NBT	▪ Routinely used for colorimetric co-distribution analysis of two mRNAs ▪ Overlap of two mRNA distributions may be difficult to visualize at cellular resolution (for this purpose sectioning may be required) ▪ Combination of fluorescent and chromogenic detection is possible ▪ Permits long-term storage of stained embryos