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Immuhistochemistry on splayed worms

Fixation

Methanol fixation is the easiest and quickest, and especially on embryos gives good morphology after staining. For gonads however, formaldehyde fixation gives better results.

  1. Disect gonads and/or eggs from ~20 worms placed on a subbed or poly-L-lysine coated slide in a 15 µl drop of water or egg-salts buffer. Water results in more eggs sticking to the slide, especially with poly-L-lysine coated slides, while egg-salts result in better gonad morphology.
  2. Dip the slides into liquid nitrogen. The quickest freezing possible is necessary to preserve the morphology of structures such as spindles.
  3. Crack off the coverslip.
  4. Fix 5 minutes in -20°C methanol.
  5. Fix 20 minutes in either -20°C acetone or in formaldehyde. Especially for gonads, formaldehyde fixation results in better morphology than acetone.

Staining

  1. Rinse slides in PBST.
  2. Cover the worms in 50 µl blocking solution (1% BSA and 10% of Normal Serum from the organism the secondary was generated in, in PBST).
  3. Block O/N at 4°C or for 1 hr at room temp.
  4. Wick off the blocking solution and add 50 µl of primary antibodies diluted in blocking solution.
  5. Incubate O/N at 4°C.
  6. Wash slides in Coplin jars with PBST 4 x for 15 min.
  7. Remove wash solution from around specimens with a kimwipe.
  8. Add 50 µl of secondary antibodies diluted in blocking solution.
  9. Incubate 4-5 hours at 4°C.
  10. Wash as above.
  11. For dapi staining, use either mounting medium with Dapi, or add your own solution of 1µg/ml for 30 minutes after last wash.
  12. Do a final wash with 10mM Tris pH 7.4 to 8.0 to get the salt out.
  13. Add a drop of mounting medium and put a coverslip on.
Formaldehyde fixative
0.5 M Hepes, pH 6.9 32 ml
0.5 M EGTA 0.312 ml
1M MgSO4 0.320 ml
37% Formaldehyde 20 ml
10x PBS 20 ml
H2O to 200 ml