1. Wash animals in M9 buffer; slowly apply 200 µl of worms to moistened 7mm X 10mm rectangles of Millipore HA filter paper (0.45 micron pore size; this filter will dissolve in acetone) while paper rests on glass fiber filtration unit under weak vacuum and add 0.5 ml of M9. Immediately place this onto wet Whatman filter paper that is positioned on styrofoam freezing disk supplied with the Reichert MM80E slam freezer.
2. Attach paper to plunger and slam worms against cold copper block (in liquid nitrogen). Transfer freezing disk and worms into dewar of liquid nitrogen.
Freeze Substitution
3. Transfer specimen into mesh basket while submerged in liquid N2. Hold frozen sample at ñ90°C for 48 hours or more in primary freeze substitution medium: 1.0% OsO4 in acetone
4. Warm sample to ñ60°C in same fixative over 16 hour period
5. Warm sample to ñ 30°C in same fixative over 4 hour period
6. Warm sample to 0°C in same fixative over 1 hour period
7. Transfer sample to 100% acetone two times at room temp, 10 min
Infiltration
First change is in 1 part resin to 1 part acetone, allow to sit 30 min
Second change is in 2 parts resin to 1 part acetone, allow to sit for 30 min
Third change is into pure resin and hold one hour
Fourth change is into pure resin and hold overnight
The next day, place samples in fresh resin at room temp and place infiltrated worms between Aclar sheets.
Excess sample is embedded in eppendorf tubes
Cure samples in 60°C oven for 3 days
Cut best worms out of thin embedment and re-embed in resin and re-cure
Resin formula
Use pre-dessicated plastic bottles and mix
Solution A
80 g LX112
103 g DDSA
Solution B
100 g LX112
86 g NMA
Weigh each of the above solutions dirrectly into a bottle and violently shake to mix.
These stock solutions are stored at 4°C, and brought to room temperature before mixing the final resin, then finally adding DMP-30.
13 g soln. A
17 g soln. B
mix, mix, mix!!
then add 0.45 ml DMP-30
