Fluorescent in situ hybridization
To carry out fluorescent in situ hybridization, use the standard in situ protocol with the following modifications:
- 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.
- 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.
- 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.
- 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.
- 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!]
- 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.
- Incubate the specimens in Ab block solution on a rocking table for 1-2 hrs at room temperature.
- 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.
- Following the standard antibody wash, proceed to another TSA reaction, using the second fluorophore.
- 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.