Standard Immersion Fixation for Structure
by Hall, Hartwieg and Nguyen


Summary protocol

  1. Immerse live worms in 0.8% glutaraldehyde + 0.8% osmium tetroxide in 0.1M sodium cacodylate buffer, pH 7.4, rt.
  2. Use a scalpel blade to cut worm open in fix under dissecting scope in the hood.
  3. Move samples in petri dish onto ice and cover for 1 h.
  4. Rinse several times in 0.1M cacodylate buffer at 4°C.
  5. Fix again in 2% OsO4 in 0.1M cacodylate buffer, pH 7.4, holding at 4°C overnight.
  6. Rinse several times in 0.1M cacodylate buffer at 4°C.
  7. Embed worm pieces in 2% agarose, allowing to cure at 4°C for 20-30 min.
  8. Dehydration schedule is done at rt: 50% ethanol 10 min; 70% ethanol, 10 min; 90% ethanol, 10 min; 3X 100% ethanol, 10 min each; 50:50 ethanol/propylene oxide (PO), 15 min; 2X 100% PO, 15 min each. 
  9. Infiltration schedule done at rt on rotator or with an embedding machine, 3 parts PO to 1 part resin, 45 min; 1 part PO to 3 parts resin, 45 min; 3 changes in 100% resin over 4 h, 4 h and 8 h, respectively.
  10. Arrange samples in flat embedding mold and cure at 60°C for 2 days.

Solutions

Live worms are washed off the culture plate with 1 mL of the first fixative and immediately placed in a new Petri dish at room temperature. Using a scalpel blade, individual worms are cut open. Placement of the cut depends on the tissue of interest. Cutting should be done as quickly as possible, after which the specimens are placed on ice in the dark. Wash off fixative with cold cacodylate buffer, and immerse specimens with the second fixative, keeping them overnight in the refrigerator. Rinse again with cold buffer. Move the best worm pieces into a very small drop of buffer in a Petri dish, then withdraw most of the buffer and quickly place a drop of liquid 2% agarose over the tissue pieces. Using a hot platinum wire (a wormpick), quickly rearrange the tissue pieces side by side before the agarose can gel. Place Petri dish into refrigerator for about 10 min to firm the agarose. Turn the agarose block upside down and place another drop of the liquid agarose over the exposed worm pieces and place the dish back into the refrigerator*. After about 10 min the agarose will become firm enough to cut out a small square block in which the tissue is positioned in the center of the agarose. Begin dehydration and infiltration. Several different commercial ‘‘Epon-like’’ resins are available; any can work well, but do not try to mix two different brands. If the purchase of an embedding machine (Leica TP1020 or EMS Lynx II) is feasible, all changes of solution can be programmed in advance and an infiltration can be processed overnight without human help. This may deliver more consistent results, and ultimately save on labor. More important, these machines keep the tissue constantly moving, a key feature in guaranteeing the proper infiltration of the resin.

Helpful Hints

If using Petri dishes, the most efficient means to change solutions is to use a micropipette to move animals out of one solution and into the next in a new dish. Alternately, using a 9-well Pyrex dish, worm pieces will settle to the bottom, and old solutions can be removed from the top of the well using a 2 mL pipette without disturbing the fixed samples on the bottom. Then samples can stay in the same well through the whole procedure. It is also possible to hold cut samples in miniprep tubes containing a filter, so that old solutions are removed by suction, leaving worm pieces on the top of the filter, ready for immersion in the next solution.

For adult worms, not more than six pieces should be aligned within one block, in order to keep the final block face fairly small. By covering both sides of the worms with agarose (*above), the final agarose sandwich will give less trouble when placed into the embedding mold as animals are less likely to end up directly at the edge of the plastic block, and will be easier to trim.

A transparent embedding mold is preferable, since it permits better viewing of the specimen at the end of the infiltration step. Then animals can be well positioned under a dissecting microscope before curing. This will minimize problems in specimen trimming.

Troubleshooting

It is very important that in all infiltration steps the tissue is kept immersed in fluids and that the fluids are kept moving (shaker or slow rotator). Otherwise a good infiltration is not guaranteed. Other buffers can be used instead of cacodylate. HEPES is acceptable, but avoid phosphate buffer, which tends to cause precipitates and artifacts. An en bloc stain with uranyl acetate could also be added, but be careful to switch buffers to achieve pH 5.2 before that step (see Hall, 1995). With the advent of modern digital cameras, rather than film imaging, one often can achieve acceptable contrast even without an en bloc UAc stain.


References

Hall, D.H. 1995. Electron microscopy and three-dimensional image reconstruction. Methods Cell Biol. 48: 395-436. Abstract

Hall, D.H., Hartweig, E. and Nguyen, K.C.Q. 2012. Modern electron microscopy methods for C. elegans. Methods Cell Biol. 107: 93-149. Abstract


image