1. Wash animals in M9 buffer
2. Microwave at "hotspot" according this schedule: 2 min ON, 2 min OFF, 2 min ON, 2 min OFF, 2 min ON,
Refreshing ice bath between " ON" sessions to keep samples cold throughout
Fixative is 2.5% glutaraldehyde, 1.0% paraformaldehyde in 0.1M HEPES
After microwave bouts, continue soaking animals in same fix at room temp for one hour
3. Wash 3 X 10 min in 0.2M HEPES buffer
4. 1% OsO4, 0.5% Kfe(CN)6 in 0.1M HEPES pH 5.6, r.t., 60 min
5. Wash in 0.2M HEPES, 2 X 10 min; in 0.1M NaAcetate pH 5.2, 2 X 10 min
6. Stain in 1% UAc in 0.1M NaAcetate, pH 5.2, r.t., 45 min
7. Wash in 0.1M NaAcetate, 2 X 10 min; in 0.2M HEPES, 2 X 10 min
8. Embed cut pieces in 3% agarose, positioning worms into clusters; allow to harden at 4°C for 1 hour, or overnight under buffer.
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%, 5 min in 50%, 5min in 75%, 5 min in 95%, 3 X 10 min in 100% ethanol, 3 X 10 min in propylene oxide
Infiltration
First change is in 1 part resin to 2 parts propylene oxide, allow to sit 3-4 hours or more
Second change is in 2 parts resin to 1 part propylene oxide, for 3-4 hours or more, 1 part propylene oxide to 2 parts resin overnight, rotating.
The next day, place samples in fresh resin at room temp; place samples in air tight containers and agitate or rotate samples slowly during infiltration. Then change into fresh resin at least 3-4 times, 2-4 hours per change
Embed samples in fresh resin in embedding mold, and position carefully.
Cure samples in 60°C oven for 3 days
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
Special Equipment needed
Scientific microwave oven (we use a model from Ted Pella); kitchen ovens do not properly control the microwave energy pulses for this purpose.
See Paupard, Miller, Grant, Hirsh and Hall (2001) for further details on use of the microwave oven
