Before mid-stage 9, Helobdella embryos are enclosed in a vitelline membrane. The existence of vitelline prevents larger molecules such as antibody and riboprobe from entering the embryo, and thus for immunostaining and in situ hybridization, it is necessary to remove vitelline membrane first. The vitelline membrane could be removed before or after fixation. But for the best preservation of the embryo and the easiness, if processing the embryo, we remove vitelline membrane after fixation.

The vitelline membrane could be removed from the fixed embryos quite efficiently by passing the fixed embryos through a broken Pasteur pipette. After several washes in buffered saline, fixed embryos were placed in a dish with Sylgard bed. Embryos are sucked in and then expelled out of the pipette (in each stroke cycle, embryos pass through borehole twice, one inbound and one outbound). Inspect embryos visually after each stroke cycle, and remove and discard embryos with visible damage. Transfer the intact devitellinized embryos into a tube. Subject the remaining embryos that still have the vitelline membrane to a new stroke cycle. Repeat cycle until all embryos are devitellinized.

For the best result, the diameter of the borehole should be slightly larger than that of an embryo. A perfect borehole should be big enough to allow a few dozens of embryos passing through smoothly in a row, and ~50% of embryos should become vitelline-free in each cycle. To make the broken pipette, a glass pipette is pulled above the flame, and the tip is crushed with tweezers at the site where the diameter is slightly smaller than the desirable size. The borehole size can be optimized by gradually and slowing removing glass shard from the broken surface until the desirable size has been reached. There is no need to trim the borehole. A zigzag opening surface works better than a trimmed one. If the borehole becomes too large, just throw the pipette away. You are pretty much depending on luck to get a good pipette, so it will take a few pipettes before you get one. There is no special technique involved, but you need to be able to identify a good pipette when encountering one.