Embedding and Sectioning of Embryos

  1. Stain embryos according to standard protocols. Staining for sections should be much darker than for whole mounts--background simply will not be a problem. Do not attempt to embed embryos stained with X-gal, X-phosphate, or other ethanol-soluble chromogen.
  2. (DAY 1) Dehydrate embryos by successive 5 min. washes in 30% EtOH, 50% EtOH, 70% EtOH, and 2 or 3 changes of 100% EtOH.
  3. While embryos are dehydrating, prepare Spurr Low-Viscosity Embedding Media. Polysciences, Inc. sells a kit (their cat. # 01916) with all four components of Spurr and detailed instructions for optimizing the formulation. We simply use the standard ("firm") mix: Stir thoroughly. Store in a sealed container with dessicant; storage at -20°C increases the lifetime of the mixture.
  4. When embryos are thoroughly dehydrated, remove EtOH and add a 1:1 mixture of Spurr and propylene oxide (Aldrich Chemical Co., cat. # 24,039-7). (PO makes Spurr much less viscous, and thus more penetrant, and protects it from residual water in the sample.) Rock in this mixture overnight.
  5. (DAY 2) Replace Spurr/propylene oxide with 100% Spurr and rock for at least half an hour. Place embryos in 100% Spurr in an embedding mold (we use Polysciences cat. # 02615), and align under a stereomicroscope. Some people use Dumont forceps for aligning embryos, but I've had the best results with a finely pulled glass capillary. Be careful, as the embryos are very fragile and brittle when saturated with Spurr. My little sketch shows the correct orientation to make transverse sections:
  6. After aligning embryos as thoroughly as possible (do align them with the most-convex surface up, since they'll move around less) incubate at 65°C overnight. You may have to try several ovens before you find one that vibrates minimally enough to make this work, since the embryos can drift badly out of alignment. (This will be much worse if they're not lying on the bottom of the mold.)
  7. (DAY 3) Examine your blocks and choose the ones you'll want to section. To cut them down to size (approx. 1/3 of total length), stick a razor blade into the block parallel to the plane of sectioning. Holding the block with forceps (hold the end containing the embryos, as the unheld end may be hard to find), tap the razor blade with a hammer to cleave the block. Use a file to make the cleaved surface smooth, flat, and parallel to the opposing surface (and the desired plane of sectioning). Using a bit of Spurr (the stuff you made two days ago should still be good), glue the filed surface to a block holder (this is the thing that mounts on the microtome--we use Polysciences cat. # 15899). Once again, incubate at 65°C overnight.
  8. (DAY 4) Sectioning Day! Due to the hardness and toughness of epoxy as an embedding material, you will need a microtome with a tungsten carbide blade. (We use the TCB-80 from Energy Beam Sciences.) After adjusting your microtome as usual, cut through the block in 15 micron slices (these will curl up something fierce, but you only need to be able to tell if you've hit the embryos yet) until you want to begin collecting sections. Then cut 5 micron sections, remove them from the blade with Dumont forceps, and lay them on droplets of water (I use 4-5 microliters per drop) on a Fisherbrand Superfrost/Plus slide (Fisher cat. # 12-550-15).
  9. Lay the slide on a slide warmer (a heating block upside-down works well) at 80°C. IMPORTANT: The droplets of water MUST NOT DRY before the slide is placed at 80°C--if they do, the sections will not adhere at all. The water should be evaporated by the heat, at the same time that the sections are affixed to the slide.
  10. Examine and photograph sections with immersion oil placed directly on them--this will render the nearly-opaque resin nearly-invisible. We've found that placing oil at the edge of a section and letting it penetrate gives better results than dropping oil directly on a section, which tends to trap air in tiny grooves in the cut surface of the resin. Note that you can't use a coverslip with this resin since the immersion oil must be in direct contact with the section. Note also that the immersion oil will gradually loosen the sections from the slide, so any section to which you add oil is not archival; get as many pictures as you want now.

Steve Gisselbrecht
Michelson Lab