Fluorescent in situ hybridization

To carry out fluorescent in situ hybridization, use the standard in situ protocol with the following modifications:

  1. If multiple genes are to be probed, add a set of differently labeled probes (digoxigenin, biotin, DNP or fluorescein, each for a specific gene - see probe synthesis protocol for making probes). Digoxigenin labeling is recommended for the weakest probe. DNP is the second choice. Biotin the third, and fluorescein the fourth. If only a single gene is to be probed, follow the standard protocol.
  2. Note that a higher probe concentration than that used in the standard AP-chromogenic reaction may be needed for generating a detectable fluorescence signal. Start with ~5 ng/μL (or 5X concentration of probe as used in the standard hybridization reaction that will use BM Purple or NBT/BCIP color reaction for staining), decrease concentration if the background is too high and increase concentration if the signal is too low.
  3. DS-PreHyb is used in place of regular PreHyb. Dextran sulfate and accelerator are added to TSA reaction. These will enhance the sensitivity of probe detection.
  4. In the antibody detection step, use peroxidase-conjugated antibody (or unconjugated anti-hapten antibodies) in place of alkaline phosphatase-conjugated antibody. For Roche POD-conjugated anti-digoxigenin, add 1 μL antibody to 500 μL blocking solution.
  5. Following the antibody wash, proceed to TSA reaction. Add DS and 4-iodophenol to the TSA reaction to enhance the signal. [Sensitivity is really an issue here, do everything you can to enhance the signal!]
  6. If multiplex in situ hybridization is performed, strip the first peroxidase-conjugated antibody by washing specimens twice in acid glycine buffer, 5 min each, at room temperature. Wash the treated embryo with PBS + 0.1% Tween (PBT) for 3 times, 5 mins each.
  7. Incubate the specimens in Ab block solution on a rocking table for 1-2 hrs at room temperature.
  8. Add peroxidase-conjugated antibody against the second hapten (i.e. anti-DNP, anti-biotin, or anti-fluorescein). Incubate overnight on a rocking table at 4°C.
  9. Following the standard antibody wash, proceed to another TSA reaction, using the second fluorophore.
  10. Treat the embryo again with acid glycine buffer as in step 4. Repeat antibody labeling and TSA reaction for the third and fourth probe, if any.


Recipes

  • For cryoprotection, all antibodies used here are diluted with glycerol at an 1:1 ratio after reconstituted in buffer solution.
  • acid glycine buffer: 0.1 M glycine (7.5 g/L), pH 2, supplemented with 0.1% Tween-20.
  • Ab block solution (for blocking step of the second antibody incubation): 10% Roche Western Block Solution (without serum), 0.1% Tween-20 in PBS.