Appendix 13: Fixation Procedures

In general, the fixation procedures used will depend on the type of data desired. Because of the small size of the eggs, we suggest the use of Spurr's low viscosity embedding medium (Spurr, 1969).

Aldehyde fixation for electron microscopy

Method A: This recipe (Eckberg, 1981b; Anderson and Eckberg, 1983) is a modified Karnovsky's fixative (Karnovsky, 1965). It is good for studies of fertilization and most aspects of embryo structure, but it extracts carbohydrates.

4% paraformaldehyde, 5% glutaraldehyde, 100 mM sodium cacodylate, pH 7.8 in artificial seawater (ASW; Appendix 4)

  1. Fix 20 min at room temperature.
  2. Wash 2 h or longer at 2-4°C in ASW.
  3. Postfix 1 h at 2-4°C in 2% OsO4 in ASW.

Method B: This method (Jeffery, 1985) also gives good preservation of egg structure and extracts less carbohydrate.

2% glutaraldehyde, 1% tannic acid, 275 mM sucrose, 200 mM sodium cacodylate, pH 7.4

  1. Fix 2 h on ice.
  2. Wash three times with 200 mM sodium cacodylate, 370 mM sucrose.
  3. Postfix on ice 1 h with 2% OsO4 in 1.25% NaHCO3, pH 7.2.

Petrunkewitsch's fixative for in situ hybridization (Jeffery and Wilson, 1983)

This procedure quantitatively preserves poly(A) in Chaetopterus eggs.

Solution A: 12% HNO3, 8% Cu(NO3)2 in water

Solution B: 76% ethanol, 5.7% ethyl ether, 3.8% crystalline phenol in water

  1. Mix 1 part Solution A with 3 parts Solution B.
  2. Fix 30 min at -20°C.
  3. Dehydrate through ethanol.
  4. Clear in toluene at -20°C.
  5. Embed in paraplast.