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Mark A. Sanders

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Gilbert G. Ahlstrand
Tracy E. Anderson
Gail Celio

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Chemi Banari
Kaitlyn Bissonnette
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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

 
  Home > FAQ & Protocols > Preparation of Cryosamples

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.

 
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