About the Center

Location and Directions

Data Management System

Contact Information:

Director
Mark A. Sanders

Staff
Gilbert G. Ahlstrand
Tracy E. Anderson
Gail Celio

Students
Chemi Banari
Kaitlyn Bissonnette
Alex Cramer
Lizzy Watkins

University of Minnesota
Imaging Center
Rooms 23-35 Snyder Hall
1475 Gortner Avenue.
University of Minnesota,
St. Paul, MN 55108

Hours: 9:00-5:00 weekdays
Closed holidays

Embedding of Yeast in Spurr Resin

Fixation

1. Filter 10 OD units of cells (~1 x 10⁸ cells) on a 0.45 mm Millipore filter unit without letting the cells dry.
   
   
2. Wash on the filter with 10ml of 0.1M sodium cacodylate buffer (pH 6.8).
   
   
3. 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

1. Wash twice in 10ml of 50mM KPO4 pH 7.5, each time centrifuging the cells @ 2,000 RPM @ 4°C.
   
   
2. 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.
   
   
3. Stop zymolase digestion by centrifuging cells @ 2,000 RPM for 5 min @ 4°C.
   

Postfixation

1. 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.
   
   
2. Pellet the cells @ 14,000 RPM for 2 min @ RT.
   
   
3. 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.
   
   
4. Wash 3 x 5 minutes with water.
   

En Bloc Staining

1. Add 1ml of 2% aqueous uranyl acetate and incubate for 1 hour @ RT in the dark.
   
   
2. Wash 2 x 5 min with water.
   

Dehydration

1. 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
   
   
2. Wash 4 more times with 100% ethanol from a fresh bottle, 5 min each.
   
   
3. Wash with 100% acetone for 5 min.
   

Infiltration

1. Add 50% acetone / 50% Spurr and incubate for 2 hours on wheel. Change to 100% Spurr and incubate overnight.
   
   
2. Change Spurr 2 x over the next day. Bake @ 80°C for at least 24 hours.