1. A stock solution of fluorescent phalloidin/phallacidin (Molecular Probes) is dissolved in methanol (or DMSO, see Note 2 below) at 200 U/ml.
2. The first step of making the working solution is to pipet 1/50 working volume of methanol stock (for example, 2 μL of methanol stock for making 100 μL working solution) into a microfuge tube and allow the methanol to dry up completely. It is very important to remove methanol completely because the presence of even just a trace amount of methanol (or ethanol) would impair phalloidin-microfilament interaction. Keep the lid open to allow methanol escaping, and keep the tube in dark to reduce fluorescence photobleaching. Evaporation of methanol can be monitored by checking under a dissecting microscope once for a while. Crystallized precipitate would show up once methanol residue has gone completely. It should take less than 1 hour to dry 2 μL methanol stock in a 1.5 mL microfuge tube. Drying can be sped up by placing the tube in the vacuum or by SpeedVac.
3. To reconstitute working solution, add 50X of the initial stock volume of PBS (100 μL of PBS, if 2 μL of methanol stock was used in the beginning) to the dried precipitate or DMSO stock (see Note 2 below). Mix well. Note that phalloidin/phallacidin has a reduced stability in water solution. Always use freshly prepare working solution for staining.
4. Add the phalloidin/phallacidin working solution to the devitellinized, formaldehyde-fixed embryos. Incubate at room temperature for 30 minutes. Longer incubation does not help and may have a negative effect on the staining.
5. Wash with PBS for a few times.
6. Mount the specimen in aqueous solution for observation. We have tested early-stage leech embryos labeled with Alexa Fluor 488-conjugated phalloidin (Molecular Probes A12379) in a variety of clearing solutions, e.g. buffered glycerol, SeeDB, Scale U2, and Clear^T2. All yielded viable staining. However, staining survives relatively well in SeeDB. The signal remains detectable after weeks in SeeDB. In contrast, staining only lasts for a few hours in Scale U2 and Clear^T2. Staining also lost gradually in buffered glycerol, and the higher the concentration of glycerol is the faster the staining fades away. The loss of staining in these clearing solution is likely caused by the weakening of phalloidin association to the microfilament.

Note 1: If the specimen is to be viewed in simple salt solution (e.g. PBS), the protocol can be further simplified. In this case, staining solution is made up by adding 1/50 volume of methanol stock into 1 volume of PBS, without removing methanol. However, if glycerol or other non-water aqueous solvent is intended as clearing medium, complete removal of methanol is necessary for a successful phalloidin staining of Helobdella embryo.

Note 2: To avoid the problem with methanol, one can dissolve phalloidin/phallacidin stock in DMSO instead of methanol. In DMSO is used as a solvent, 1/50 volume of DMSO stock can be added to 1 volume of PBS to make the working solution. However, methanol stock is still preferred for long-term storage.