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

Preparation of Cryosamples

Fixation

1. Fix cells/tissue in 4% paraformaldehyde in 0.25 M Hepes pH 7.4 for 1 hour at room temperature.
   
   
2. Replace fixative with 8% paraformaldehyde in 0.25 M Hepes pH 7.4 and leave overnight @ 4°C.
   
   
3. Wash 3 times in PBS. In case of cell monolayers, scrape the cells with a rubber policeman and pellet them @ 5,000 - 10,000 RPM for 5 min @ RT.
   

Embedding in Gelatin

1. Melt gelatin (10% in PBS) by warming it up in a waterbath at 45°C.
   
   
2. Resuspend pellet in 0.5 ml of gelatin. Spin immediately @ RT, then put on ice.
   
   
3. Let gelatin set completely for 1 h on ice.
   
   
4. Using a wooden stick, loosen the block of gelatin from the tube and transfer to a dish on ice.
   
   
5. Using a razorblade or scalpel, cut the tip of the pellet into small pieces.
   

Infiltration with Sucrose

1. Transfer pieces into an Eppendorf tube containing 2.3 M sucrose in PBS.
   
   
2. Gently swirl the bottle and leave overnight at 4°C.
   

Freezing Down Samples

1. Transfer pieces of pellet/tissue onto plastic nail-holder making sure to cover them completely with sucrose. Dissect them into smaller pieces using a razorblade.
   
   
2. Transfer the small pieces onto aluminum nails/studs, leaving just enough sucrose to “glue” them to the nail.
   
   
3. Immediately plunge the nail into liquid nitrogen using the special orange forceps.
   
   
4. Transfer the nails into a cryotube (Nunc) and store in liquid nitrogen.