Original Protocol for Fluorescent RNA In-situ using Tyramide Signal ...

Gavin WilkieIlan Davis Lab, Biochemistry Prehybridise at least 1 hour in Hybe buffer at 70°C (on a heat block or in an oven) 5. Hybridisation Add Dig-­‐RNA probe in Hybe (Usually diluted 1:1000, but test dilution for optimal results) to embryos using at least 100µl of probe making sure embryos are well covered and mixed with the probe. Leave overnight at 70°C (on a heat block or in an oven) 6. Washes: In morning remove and save probe (it can be used several times but does get diluted each time). Wash as follows (at 70°C, warm up solutions first; no need to agitate): 1 X 30 min Hybe 1 X 30 min 50% Hybe / 50% PTW 4 X 30 min PTW BLOCKING STEP (not normally required) incubate in TNB for 1 hour 7. Antibody: Incubate embryos in 1:1000 Sheep anti-­‐Dig POD (Horse Radish Peroxidase) coupled antibody (Boehringer Mannheim Cat no. 1 207 733) in PTW. Rock the tubes on a rolling incubator for at least 1 hour. NB Can include fluorescently labelled Wheat Germ Agglutinin (Molecular Probes, 1/1000) during this step or at the end of the procedure to stain nuclear membranes (binds to NPC’s). 8. Washes: Remove antibody (can save and reuse) Wash 3 X 20 min with PTW. 9. TSA Detection Before going on to do the Tyramide Signal Enhancement, it is a good idea to check the in-­‐situ has worked using the DAB substrate for HRP. Remove a small amount of embryos to a microtitre plate and stain with DAB. The signal should be visible within minutes. The TSA signal is not visible, so staining intensity can only be controlled by staining for different lengths of time and evaluating the sample in the fluorescent microscope. However, the time taken to see a good DAB signal is roughly equivalent to the time for a good TSA signal. For each sample, Dilute 2μl of the Fluorescent Tyramide stock solution (NEN) into 100μl of Amplification Diluent (supplied with kit). Remove as much PTW from the embryos as possible and add the TSA. Incubate for 1-­‐5 mins depending on level of staining required. 10. Stop reaction by washing 3 x 5 mins with PTW. Can then stain with other antibodies (e.g. anti-­‐tubulin monoclonal, or Wheat Germ Agglutinin) It is often useful to stain with DAPI at this point -­‐ Use 10 times the normal amount (e.g. 1/1000 dilution of 1mg/ml stock)