For Helobdella embryos, most chromogenic substrates are not sensitive enough for whole-mount in situ hybridization. The only combination that might work is BM Purple (or NBT/BCIP, both purple) and INT/BCIP (orange). Note that INT/BCIP is soluble in ethanol or other organic solvents, thus it would not be possible to embed the sample for a section (though one might consider frozen section). Fluorescent multiplex in situ hybridization is probably more useful than this method if the transcripts are expressed within the detection limit of the HRP-TSA method. Nonetheless, if the gene expression levels are low and the embryological context is relatively simple (e.g. stage 8 and earlier), one might consider using this protocol instead.

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

1. In day 2, add the two differently tagged probes (digoxigenin, biotin, DNP or fluorescein - see probe synthesis protocol for making probes) each at the optimal concentration as empirically determined in single-probe in situ hybridization. Digoxigenin labeling is recommended for the weaker probe.
2. In day 3/4 (antibody detection), add the antibody against the weak probe at an appropriate dilution (i.e. AP-conjugated anti-digoxigenin, 1:2500).
3. Following the antibody wash, proceed to coloration reaction with either BM Purple or NBT/BCIP.
4. Strip the first AP-conjugated antibody by treating specimens with acid glycine buffer for 10 min at room temperature. Wash the treated embryo briefly with PBS + 0.1% Tween (PBT) for 3 times. Then followed by 3X 5-min PBT washes.
5. Incubate the specimens in Ab block solution on a rocking table for 1 hrs at room temperature.
6. Add AP-conjugated antibody against the second hapten (i.e. AP conjugated anti-biotin, anti-DNP or anti-fluorescein at 1:500) or unconjugated primary antibody against the second hapten (anti-biotin, anti-DNP or anti-fluorescein, 1:500) Incubate overnight on a rocking table at 4°C. Since most commercial anti-biotin, anti-DNP and anti-fluorescein antibodies are produced in common species such as mouse and rabbit, we simply use the unconjugated primary antibodies for these haptens and use the alkaline phosphatase-conjugated secondary antibody for detection. In this case, do a secondary antibody incubation (i.e. goat anti-mouse AP conjugate, 1:500) after washing off excessive primary antibody as in regular immunostaining.
7. Following the standard antibody wash, proceed to INT/BCIP color reaction.
8. Treat the embryo again with acid glycine buffer for 10 min at room temperature. Acid treatment seems to retard photochemical reaction that turns the yolk tissue into dark color in a glycerol solution.
9. Transfer the embryo into PBS-glycerol solution for photographing. Take photos as soon as the glycerol infiltration is completed (as the embryos sink to the bottom of the tube) since the yolky tissue will turn dark after prolonged exposure to glycerol and light despite the acid treatment. INT/BCIP is soluble in ethanol, and thus the double-stained specimen should be viewed in buffer glycerol.

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 (and 5% normal goat serum, if goat anti-mouse/rabbit AP conjugated secondary antibody is used), 0.1% Tween-20 in PBS