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 |