Fixation and storage of stickleback embryos and fry for in situ hybridization

Mike Shapiro

(version 1.0;   01/27/04)

 

This fixation method can be used for in situs on whole-mounts or sections (cryostat or paraffin) in most vertebrate embryos, and also should work well for some immunohistochemistry protocols.

 

Prepare 500 ml of 4% paraformaldehyde fixative

 

  1. Prepare 500 ml of 1X PBS, pH 7.4, using MilliQ-filtered or other high-quality water. Filter through 0.2 µm filter and autoclave. Alternatively, use pre-packaged, sterile PBS solution, such as Gibco cat. # 10010-023.
  2. Combine PBS and 20 g paraformaldeyde powder (Sigma cat. # P-6148) in a 1 L flask with clean stir bar. Paraformaldehyde is highly toxic and should only be handled in a fume hood.
  3. Stir mixture while applying heat. The powder will dissolve very slowly at first, but more quickly as the temperature approaches 65°C. Do not allow temperature to exceed 65°C.
  4. When the temperature reaches 65°C, turn off the heat and stirrer. There may still be a small amount of undissolved powder at the bottom of the flask. Allow the undissolved powder to sink to the bottom of the flask, and decant the solution into 50-mL concial tubes. The solution will expand when frozen, so decant no more than 40 mL per tube. Alternatively, the solution may be run though a sterile 0.2 µm filter and then aliquotted into 50 mL tubes.
  5. If solution is to be used immediately, place tube(s) on ice. Otherwise, store the tubes at ­20°C for up to a year. Once the solution is thawed, use within 24 hours.

 

Fixation of specimens

 

  1. Thaw frozen 4% paraformaldehyde (4% PFA). Solution can be heated in water bath up to 65°C to dissolve precipitate.
  2. Collect specimens in a 50 mL conical tube (in tank water).  Do not to exceed about 1 mL of fish per tube.
  3. Drain tank water from tube. If fish are likely to swim out, place a strainer at the end of the tube. Netted inserts for 6-well cell culture plates (Corning cat. # 3480) work well for this purpose.
  4. Add „25 mL of ice-cold 4% PFA to the tube. Fix specimens for 24 hours at 4°C. Placing the tube on a rocker will help with fixation.
  5. Decant 4% PFA and wash specimens three times, 7 minutes per wash, in 40 mL sterile 1X PBS. Use a rocker during washes, if available.
  6. Decant final PBS wash and add 25 mL 100% methanol. Invert tube several times and allow specimens to sink to bottom of tube.
  7. Decant and add 25 mL fresh 100% methanol.
  8. Store specimens at ­20°C for up to 1 year. Note that, unlike dehydration, re-hydration for in situs or sectioning should be done incrementally through a MeOH:PBS series (MeOH -> 3:1 -> 1:1 -> 1:3 -> PBS, 5-10 min per step).