turboFISH
Normally, when you do RNA FISH, you need to do an overnight hybridization. Using the standard protocol, you can reduce this to around 4-10 hours or so, but that's about as fast as it goes. Sydney Shaffer (graduate student in the lab), however, has introduced a modified protocol in which you can do RNA FISH hybridizations in just 5 minutes, sometimes as fast as 30 seconds! The key is to use an alcohol fixative rather than a cross-linking fixative while increase probe concentration. And then... magic. Also, while you do have to use a higher concentration of probe, you also use a smaller volume, so you don't even burn that much extra material. We have just published the protocol in PLoS ONE, and below is a nice and detailed protocol.
Also, here's a video:
turboFISH protocol
Prepare wash buffer and hybridization buffer (same as usual protocol):
Wash buffer: 2X SSC with 10% formamide
Hybridization buffer: 2X SSC with 10% dextran sulfate and 10% formamide
Turbo hybridization:
- Put methanol (or ethanol) in -20C freezer for 30 minutes prior to the experiment.
- Put wash buffer into the media warmer at 37C.
- Wash cells twice with 1X PBS (we use 1mL per wash with a 2-well chambered coverglass).
- Add 1mL of -20C methanol (or ethanol) to each well of the chambered cover glasses.
- Put entire dish containing samples into the -20C for 10 minutes.
- While cells are fixing, prepare hybridization buffer:
- 1µL of each probe (at around 3.56mM, which is typically about 20x to 40x the usual RNA FISH probe concentration
- 1µL DAPI (50µg/mL)
- 50µL 10% hybridization buffer
- When the time is up, remove the sample from the freezer.
- Aspirate the methanol or ethanol from the sample
- Add 5uL hybridization buffer with probe and DAPI to the center of well.
- Carefully cover the solution and your sample with a coverslip. The coverslip should distribute the fluid evenly across the sample.
- Place sample on the hotplate at 37C for 5 minutes.
- When the time is up, remove from hot plate.
- Add 1mL of pre-warmed wash buffer to the well and remove the coverslip from the top of the sample (we use tweezers with a hook to grab the edge of the coverslip for removal).
- Wash 3 times (generously) with wash buffer and incubate on the hot plate for 1 minute with each wash.
- Aspirate off wash buffer. Wash once with 2X SSC.
- Add 1mL of 2x SSC to the cells and view under the microscope.
- Enjoy your rapidly acquired and beautiful images!