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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 live demo


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:

  1. Put methanol (or ethanol) in -20C freezer for 30 minutes prior to the experiment.
  2. Put wash buffer into the media warmer at 37C.
  3. Wash cells twice with 1X PBS (we use 1mL per wash with a 2-well chambered coverglass).
  4. Add 1mL of -20C methanol (or ethanol) to each well of the chambered cover glasses.
  5. Put entire dish containing samples into the -20C for 10 minutes.
  6. While cells are fixing, prepare hybridization buffer:
    1. 1µL of each probe (at around 3.56mM, which is typically about 20x to 40x the usual RNA FISH probe concentration
    2. 1µL DAPI (50µg/mL)
    3. 50µL 10% hybridization buffer
  7. When the time is up, remove the sample from the freezer.
  8. Aspirate the methanol or ethanol from the sample
  9. Add 5uL hybridization buffer with probe and DAPI to the center of well.
  10. Carefully cover the solution and your sample with a coverslip.  The coverslip should distribute the fluid evenly across the sample.
  11. Place sample on the hotplate at 37C for 5 minutes.
  12. When the time is up, remove from hot plate.
  13. 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).
  14. Wash 3 times (generously) with wash buffer and incubate on the hot plate for 1 minute with each wash.
  15. Aspirate off wash buffer. Wash once with 2X SSC.
  16. Add 1mL of 2x SSC to the cells and view under the microscope.
  17. Enjoy your rapidly acquired and beautiful images!
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