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
Alex Cramer

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 Cells in Suspension

Fixation

1. Spin the cells at 2,000g for 5 min at 4°C and remove medium.
   
   
2. Add 2.5% gluteraldehyde in 0.1M sodium cacodylate buffer (pH 7.4) and fix for 1 hour @ RT.
   
   
3. Wash cells with 0.1M sodium cacodylate buffer 3 x 5 minutes. Each time spin the cells at 2,000g for 5 min, RT.
   
   
4. After final wash, spin the cells at 5,000 - 10,000 RPM in an Eppendorf centrifuge, 5 min at RT.
   

Post Fixation

1. Postfix cells in 1% osmium tetroxide in 0.1M cacodylate buffer for 1 hour @ RT in the hood (dislodge the pellet from de wall of the tube with needle to optimize penetration of osmium).
   

En Bloc Staining

1. Wash in 50mM sodium maleate buffer (pH 5.2) 3 x 5 minutes.
   
   
2. Stain in 2% uranyl acetate in maleate buffer for 1 hour @ RT in the dark.
   

Dehydration

1. Wash in water 3 x 5 minutes.
   
   
2. Transfer pellets into glass vials.
   
   
3. Dehydrate pellets 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 10 minutes
   
   
4. Replace ethanol with propylene oxide
   
   
5. Leave in fresh propylene oxide for 10 minutes with the lid closed.
   

Infiltration

1. Replace with 50% propylene oxide / 50% Epon mix. Leave on the wheel for 2 hours with the lid closed.
   
   
2. Replace with pure Epon and leave on the wheel for 2 hours with lid open. Repeat once.
   
   
3. Transfer the cell pellets to fresh Epon in moulds or Beem capsules (remove air bubbles). Add computer-printed labels. Cure in the oven overnight @ 60°C.