Cryoprotecting Tissue

UNDER CONSTRUCTION- KP

27 Jun 2022

Sucrose in PB Solution for Cryoprotection of Brains after Perfusions

Scope: 30%/15% Sucrose in 0.1M phosphate buffer is used for the cryoprotection of brains post perfusion.

Materials:

• • Sodium Phosphate, dibasic, anhydrous (Na₂HPO₄)
  • Sodium Phosphate, monobasic, monohydrate (NaH₂PO₄ ∙ H₂O)
  • Sucrose, Ultrapure
  • MilliQ Water
  • Nutator/Agitator
  • Cold Room/Refrigerator with outlet

Methodology:

To make 500 ml of 30% Sucrose in 0.1M phosphate buffer:

• Measure out 300 ml of MilliQ water and add it to the 500 ml bottle.
• Measure out 5.96 g of Na₂HPO₄ (sodium phosphate dibasic, anhydrous) in a weigh boat, add to the bottle
• Measure 0.803 g of NaH₂PO₄ (sodium phosphate monobasic, monohydrate) in a weigh boat, add to the bottle.
• Allow for solids to completely dissolve before adding MilliQ water to bring the volume to 400 ml.
• In a weigh boat accurately weigh 150 g of ultra pure Sucrose, record lot number in the proper reagent tracking form.
• Add the Sucrose to the 500 ml bottle.
• Stir the 30% Sucrose solution until all solids have dissolved.
• Transfer the solution to a clean graduated cylinder.
• Bring the volume of the solution up to 500 ml using MilliQ water.
• Filter the Sucrose solution through the 0.45 µm filter provided.
• Label the bottle with “30% Sucrose in 0.1M Phosphate Buffer”, the date on which it was prepared and your initials.
• Store the 30% Sucrose solution at 4ºC.

 To make 500 ml of 15% Sucrose in 0.1M phosphate buffer:

• Measure out 300 ml of MilliQ water and add it to the 500 ml bottle.
• Measure out 5.96 g of Na₂HPO₄ (sodium phosphate dibasic, anhydrous) in a weigh boat, add to the bottle
• Measure 0.803 g of NaH₂PO₄ (sodium phosphate monobasic, monohydrate) in a weigh boat, add to the bottle.
• Allow for solids to completely dissolve before adding MilliQ water to bring the volume to 400 ml.
• In a weigh boat accurately weigh 75 g of ultra pure Sucrose
• Add the Sucrose to the 500 ml bottle.
• Stir the 15% Sucrose solution until all solids have dissolved.
• Transfer the solution to a clean graduated cylinder.
• Bring the volume of the solution up to 500 ml using MilliQ water.
• Filter the Sucrose solution through the 0.45 µm filter provided.
• Label the bottle with “15% Sucrose in 0.1M Phosphate Buffer”, the date on which it was prepared and your initials.
• Store the 15% Sucrose solution at 4ºC.

Cryoprotection with Sucrose with Prepared Solutions:

• Place tissues in 15% sucrose in PBS until tissue sinks (6-12 hrs).
• Place tissues in 30% sucrose in PBS overnight or until tissue sinks.
• Best if the tissues are gently nutated at 4ºC.