Imagine you have a full week of lab work to complete and one protocol requires days of preparation and each of the downstream steps is dependent on successful completion of the preceding actions. This particular protocol is like walking a tight rope, each step requires your full concentration, and any misstep sends you back to the beginning- wasting a lot of precious time.
For some such a tedious protocol might be Fluorescence In Situ Hybridization (FISH).
FISH is a technique in which a labeled DNA or RNA probe allows one to visualize by fluorescence microscopy DNA or RNA within a cell.
This method is used to localize and assemble sequencing scaffolds into chromosomes and, to detect and localize the expression of transcripts in cells.
Sample and probe preparation for FISH, particularly for localization of RNA, are often time-consuming and laborious. Generation of an RNA probe can involve cloning an amplicon into a vector, linearizing it and then using in vitro transcription to incorporate digoxigenin or fluorescein into the probe.
Preparation of tissue samples, hybridization and detection are also multi-day processes.
At the end of many days of work, it would be encouraging to have some guarantee that your efforts will yield good results. The truth is you could spend days generating a labeled RNA probe, dechorionating embryos, preparing salivary glands or other tissues for two color FISH- only to discover in the final microscopy steps that you can not resolve your expression domains because the signals are obscured by background noise.
If this struggle sounds familiar, a paper published in this month’s issue of BioTechniques may offer some helpful tips. Schumacher et al. tested a two-color FISH protocol in zebrafish embryos combining the chromogenic substrates NBT/BCIP and Vector Red – both are alkaline phosphate substrates yielding different color products.
These substrates are not unique, but their use allowed the authors to monitor the development steps of the alkaline phosphatase reaction and maximize signal to noise ratios. By doing so, the authors were able to clearly visualize a weakly expressed transcript whose expression domain overlaps with that of more strongly expressed transcripts within the inner cell mass of zebrafish embryos.
Schumacher and colleagues also managed to generate some lovely fluorescence microscopy images in the process.
Although the technology described here is not new the improvements described by the authors could help mitigate some of the struggles associated with RNA FISH.
The protocol provided by the authors is detailed enough to allow reproduction of their method in any model organism and with any luck, it will save FISH users some precious time.
Jennifer A. Schumacher, Emma J. Zhao, Matthew J. Kofron, and Saulius Sumanas (2014) Two-color fluorescent in situ hybridization using chromogenic substrates in zebrafish. BioTechniques 57:254-256 doi 10.2144/000114229