Conventional Two Step Fixation
See Hall, 1995


Summary protocol

  1. Wash animals in M9 and anaesthetized in 8% alcohol in M9, 5 min.
  2. 2.5% glutaraldehyde, 1% paraformaldehyde in 0.1M sucrose, 0.05M cacodylate on ice, 2 h.
  3. 3 rinses in 0.2M cacodylate on ice, 10 min each.
  4. 0.5% OsO4, 0.5% KFe(CN)6 in 0.1M cacodylate, 90 min on ice.
  5. 3 buffer washes, 10 min. each, 0.1 cacodylate; 3 buffer washes, 10 min each (0.1M NaAcetate).
  6. Stain in 1% UAc in 0.1M NaAcetate, pH 5.2, rt, 60 min.
  7. 3 rinses buffer (0.1 NaAcetate) 5 min each.
  8. 3 rinses in distilled water, leave overnight, embed cut pieces in 3% seaplafue agarose

  9. Cut out small agarose blocks containing worms; should be large enough to be excluded from pipette tip, but small enough to fit into embedding mold.
  10. Dehydrate through ethanols; 5 min in 30% ETOH, in 50% ETOH, in 75% ETOH, in 95% ETOH, 3 X 10min in 100%, 3 X 10 min ethanol, 3 X 10 min in 100% propylene oxide; all at room temp. First change is in 1 part resin to 2 parts propylene oxide, allow to sit 3 h. Second change is in 2 parts resin to 1 part propylene oxide, for 5 h. Then change into fresh resin at least 3-4 times, 2-4 h per change.
  11. Place samples in fresh resin at room temp; place samples in air tight containers and agitate or rotate samples slowly. Rotation or agitation of the samples helps with the infiltration.
  12. Place samples in fresh resin in embedding mold, and position carefully.
  13. Cure samples in 60°C oven for 3 days Press straight down hard enough to immobilize the adults without rupturing most of them.
  14. Carefully (without sliding) put the slides on dry ice for at least 20 min.
  15. At this point, you can save the slides at -80°C (with possibly some decrease in staining). Let slides from -80°C ‘warm up’ on dry ice for 20 min. before cracking.

Detailed description for immersion fixation

Resin formula - use pre-dessicated plastic beaker with gradations for easy measurements
45 ml Scipoxy 812 Other brands include Embed 812, Medcast, Epon
30 ml DDSA
25 ml NMA
Mix components well with wooden applicator
Add accelerator, 1.5 ml DMP-30
Mix resin thoroughly again
Place resin into dessicator and put under vacuum for 5-15 min to remove gasses from mixture before using

Changes
This protocol includes some minor changes from the method shown in the book chapter:

  • Osmolarity of the aldehyde fix is adjusted with some added sucrose to increase it
  • Potassium ferrocyanide is added to the osmium fix to improve membrane contrast
  • Osmium tetroxide can be used at 0.5% - 2.0%
  • Same results can be obtained while fixing at each step at room temperature
  • Exact fixation and staining times are flexible
  • Since Medcast resin is no longer available, we are using either Scipoxy resin or Embed 812 resin

  • References

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


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