Immuhistochemistry on splayed worms
Methanol fixation is the easiest and quickest, and especially on embryos gives good morphology after staining. For gonads however, formaldehyde fixation gives better results.
- 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.
- Dip the slides into liquid nitrogen. The quickest freezing possible is necessary to preserve the morphology of structures such as spindles.
- Crack off the coverslip.
- Fix 5 minutes in -20°C methanol.
- Fix 20 minutes in either -20°C acetone or in formaldehyde. Especially for gonads, formaldehyde fixation results in better morphology than acetone.
- Rinse slides in PBST.
- 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).
- Block O/N at 4°C or for 1 hr at room temp.
- Wick off the blocking solution and add 50 µl of primary antibodies diluted in blocking solution.
- Incubate O/N at 4°C.
- Wash slides in Coplin jars with PBST 4 x for 15 min.
- Remove wash solution from around specimens with a kimwipe.
- Add 50 µl of secondary antibodies diluted in blocking solution.
- Incubate 4-5 hours at 4°C.
- Wash as above.
- For dapi staining, use either mounting medium with Dapi, or add your own solution of 1µg/ml for 30 minutes after last wash.
- Do a final wash with 10mM Tris pH 7.4 to 8.0 to get the salt out.
- Add a drop of mounting medium and put a coverslip on.
|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|