Specimens fixed in buffered 4% formaldehyde solution are broadly applicable to numerous downstream utilities.

For the best results, we used EM-grade 16% formaldehyde (EMS 15710 or Polysciences 18814) solution sealed in an inert glass-filled 10 mL glass ampule. Remove the liquid from the ampule using a long glass pipette, and collect the liquid in the 15 mL conical tubes. Then, divides the stock into 200 μL aliquots and keep them in -20°C freezer for long-term storage. If the formaldehyde solution can be used up within weeks, it is okay to store the solution in 4°C instead.

Salt concentration in the fixative is important to maintain the morphology of fixed specimen. For early embryos (stage 1-7), use 4% formaldehyde in 0.25X PBS or 0.5X PBS [i.e. 16% formaldehyde : 1X PBS : ddH2O = 1:1:2 (for 0.25X PBS) or 1:2:1 (for 0.5X PBS)]. For stage 8/9 embryos, use 4% formaldehyde in 0.5X PBS. For stage 10/11 embryos and juveniles, use 4% formaldehyde in 0.5X PBS or 0.75X PBS.

For leech embryos, fixation should be 1 hour at room temperature or overnight at 4°C. Over- and under-fixation tends to have a negative impact toward downstream utility. Wash with buffered saline at least three times after fixation to reduce fixative contamination in the specimen.

Formaldehyde fixation can be carried out in fixative containing <1% of Tween-20 or Triton X-100 to improve the tissue permeablization. DNA stain (DAPI or Hoechst dyes) can also be supplemented to the fixative for counterstaining. In fact, DNA counterstaining during fixation appears to produce better results than counterstaining after fixation.