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Table 1 Application of different detection systems in two-color WISH

From: Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems

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