We perform RNA ISH (mRNA or miRNA) on a high-throughput platform (a Tecan EVO liquid handling robot modified to handle slides). This is a semi-automated liquid handling system modified with a flow-through technology for slide handling and sophisticated temperature control providing automation of approximately 90% of all manual steps in the ISH protocol. All steps of ISH – from pre-hybridization to additions of probes, hybridization, stringency washes and antibody-mediated chromogenic or fluorescent reaction – are carried out by the robotic system. This gives high-quality and reproducible results. We have a 196-slide capacity per run, but most often do 40-96 slides per experiment.
We are using a dual signal amplification system including digoxigenin (DIG)- or fluorescein (FITC)-labeled probes, anti-DIG or anti-FITC horseradish peroxidase labeled antibodies and tyramide amplification. This method achieves the sensitivity of radioactivity with added cellular resolution and it can be viewed with brightfield or fluorescence microscopy.
Using a single probe [view schematic for single labeling], you can choose to develop the signal with a chromogenic method (alkaline phosphatase with BCIP/NBT, a purple precipitate) or fluorescence (Cy3, FITC or Cy5).
If you want to look at two genes at the same time [view schematic for dual labeling], we do dual ISH and develop with fluorescent systems (Cy3/FITC, Cy3/Cy5 or FITC/Cy5).
We can look at three genes at the same time, using DNP-labeled probes and suitable development systems, but find that this works less robustly than the two-gene protocol we use routinely – contact the core if you need this to discuss the feasibility for your project.
We can do ISH in combination with immunofluorescent antibody staining, but it depends on your antibody. The ISH technique uses proteinase K digestion which is detrimental to many antigens. In our experience antibodies recognizing structural proteins (for example glial fibrillary acidic protein or neurofilament H) or nuclear proteins (for example BrdU) work ok. We recommend trying your antibody on a few slides before committing to a larger number.
