Filter 10 OD units of cells (~1 x 108 cells) on a 0.45 mm Millipore filter unit without letting the cells dry.
Wash on the filter with 10ml of 0.1M sodium cacodylate buffer (pH 6.8).
Add 10ml of 3% gluteraldehyde in 0.1M sodium cacodylate buffer pH 6.8 and immediately disconnect filter unit from vacuum line. Transfer to a 15ml centrifuge tube. Continue fixation for 1 hour @ RT, then overnight @ 4°C.
Zymolase Digestion
Wash twice in 10ml of 50mM KPO4 pH 7.5, each time centrifuging the cells @ 2,000 RPM @ 4°C.
Make a 0.25mg/ml solution of zymolase in 50mM KPO4 pH 7.5. Incubate mixture @ 37°C for 15 min while shaking. Remove insoluble particles by centrifuging @ 2,000rpm for 5 min @ RT (20°C). Resuspend cells in 2ml of zymolase supernatant. Incubate tubes for 25 minutes @ 37°C while shaking.
Stop zymolase digestion by centrifuging cells @ 2,000 RPM for 5 min @ 4°C.
Postfixation
Wash 2 x in 5ml of ice-cold 0.1M sodium cacodylate pH 6.8, each time centrifuging them @ 2,000 RPM @ 4°C. Resuspend them in 1 ml of buffer and transfer to Eppendorf tube.
Pellet the cells @ 14,000 RPM for 2 min @ RT.
Add 0.5ml of cold 2% osmium tetroxide in 0.1M sodium cacodylate pH 6.8. Dislodge the pellet from the bottom of the tube using a needle to ensure good penetration of the osmium. Incubate for 1 hour on ice in the hood.
Wash 3 x 5 minutes with water.
En Bloc Staining
Add 1ml of 2% aqueous uranyl acetate and incubate for 1 hour @ RT in the dark.
Wash 2 x 5 min with water.
Dehydration
Dehydrate cells in the following order:
50% ethanol 2 x 5 minutes
70% ethanol 2 x 5 minutes
90% ethanol 2 x 5 minutes
100% ethanol 3 x 5 minutes
Wash 4 more times with 100% ethanol from a fresh bottle, 5 min each.
Wash with 100% acetone for 5 min.
Infiltration
Add 50% acetone / 50% Spurr and incubate for 2 hours on wheel. Change to 100% Spurr and incubate overnight.
Change Spurr 2 x over the next day. Bake @ 80°C for at least 24 hours.
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