The following guidelines are specific for the whole-mount preparation of Helobdella embryos.

Generally speaking, HRP reaction is fast and AP slow (HRP: minutes, AP: hours to days), but AP is more sensitive than HRP.

Horse Radish Peroxidase (HRP)

• DAB

DAB coloration reaction is not sensitive enough for whole-mount in situ hybridization of leech embryo.

DAB is suitable for immunostaining for protein antigen. However, the non-specific background is quite a problem for early-stage embryos.

• NovaRed (Vector Labs)

NovaRed is said to be more sensitive than DAB, but we have not tried it on a leech embryo whole mount yet. Stay tuned.

• Tyramide Signal Amplification (TSA) System (Perkin Elmer and Molecular Probes)

TSA works effectively and robustly for immunostaining for native protein.

Using TSA for fluorescent in situ hybridization is tricky, however. It recommended that a higher probe concentration (up to 10 ng/μl) is used. And a higher concentration of tyramide in HRP reaction may be required. The probe concentration and tyramide concentration would require optimization case by case.

Alkaline Phosphatase (AP)

• NBT/BCIP vs BM Purple

They both produce a precipitate that appears dark blue in BBBA or resin-based solution. In glycerol, the precipitate looks somewhat purplish.

Both are suitable for in situ hybridization.

BM Purple is recommended for rare or low-to-medium-level transcript (>80% of the probes tested previously belong to this category) and for stage 1-9 Helobdella embryos; staining for over-abundant transcript leads to a confusing mix of normal blue purple staining and whiteout or turquoise staining (shortening reaction does NOT help; this is caused by too fast a reaction rate; diluting probe in hybridization solution sometimes help). Color reaction for BM Purple is considerably slower than NBT/BCIP, but the contrast provided by BM Purple is significantly stronger than NBT/BCIP. However, BM Purple can generate high background in late-stage Helobdella embryos. For late-stage embryos, BM Purple background can be reduced by reducing probe concentration drastically.

For abundant transcripts, NBT/BCIP is recommended. It is also recommended for late stage embryos. (It is possible that cells elevate their transcriptional activities after assuming differentiation, and thus late-stage embryos have a higher base level transcription, making them more suitable for NBT/BCIP coloration than early embryos). NBT/BCIP works fine for transcript whose staining become visible after up to 3 hrs of color reaction at room temperature. Extending color reaction beyond 3 hours is not recommended for that it produces a higher background and hard-to-remove particle precipitations on the surface of the specimen. For such transcripts that require longer color reaction, BM Purple is recommended.

Due to the difference in reaction rate, staining of BM Purple looks somewhat more diffusing than NBT/BCIP, but this is OK for most purposes.

BM Purple is NOT recommended for immunostaining (for the same reason as in situ hybridization for abundant transcript).

Both color reaction substrates are insoluble and compatible with all forms of clearing and embedding reagents.

The precipitate is detectable with a confocal microscope:

Backscatter imaging with a long-wavelength laser (Jékely and Arendt, 2007. BioTechnique 42, 751-755); for leech embryos, specimen must be optimally stained and cleared in BBBA to achieve the best contrast;

Fluorescence: 633 nm excitation; 800-900 nm emission (Trinh et al., 2007. BioTechnique 42, 756-759).

• BCIP alone

Using BCIP alone as AP substrate produce a turquoise stain.

Lacking an oxidizing partner in color reaction, BCIP alone is significantly less sensitive than NBT/BCIP and is thus NOT recommended for in situ hybridization in leech embryos. The turquoise color does not look particular well on a leech embryo whole mount. The color contrast is low.

• INT/BCIP

INT/BCIP produces a brown/orange precipitate.

INT/BCIP has a similar sensitivity to NBT/BCIP, and its color reaction rate is comparable to NBT/BCIP. But due to its color, it will require some degree of overstaining in order to stand out visually against the pale background of embryonic tissue. It can be used as a second color for the double in situ hybridization of Helobdella embryos.

INT/BCIP precipitate is soluble in alcohol. Thus, it is not compatible with clearing agents requiring dehydration.

• Fast Red and Vector Red

They both produce a brilliant red precipitation that is so fluorescent under a regular TRITC/rhodamine/Cy3/DiI filter set.

They are NOT recommended for whole-mount in situ hybridization for Helobdella embryos due to insufficient sensitivity.

They are suitable for immunostaining and are a good secondary stain contra dark primary stain.

The stained embryo must be cleared in an aqueous solution such as glycerol because their precipitates are soluble in alcohol.