Cryoprotectant Protocols for Storage of

Free-Floating Brain Sections

 

 

 

The following protocols are used for long term storage of unstained free-floating brain sections. Transfer brain sections to the cryoprotectant and store at -20°C. Based on purpose of staining, choose the appropriate one for storing brain sections.

 

Cryoprotectant 1 (Use for Light Microscopy)

 

To make 500 ml cryoprotectant

• Sucrose ---------------------------- 150 g

• 0.1M PB ---------------------------- 200 ml

• Ethylene glycol -------------------- 150 ml

• Add more 0.1M PB to final volume of 500 ml and store at -20°C.

Cryoprotectant 2 (Use for both Electron Microscopy & Light Microscopy)

 

To make 1000 ml cryoprotectant

• Ethylene glycol ------------------- 300 ml

• Glycerol --------------------------- 300 ml

• 0.2M PB --------------------------- 100 ml

• Distilled water ------------------- 300 ml

Mix well and store at -20°C.

 

Cryoprotectant 3 (Use for Immunofluorescence)

 

To make 1000 ml cryoprotectant

• Sucrose ----------------------------- 300 g

• Polyvinyl-pyrrolidone (PVP-40) --- 10 g

• 0.1M PB ----------------------------- 500 ml

• Ethylene glycol --------------------- 300 ml

Add PVP-40 to 0.1M PB. Stir to dissolve. Slowly add the sucrose to dissolve, and then add the ethylene glycol and bring the final volume to 1000 ml with 0.1M PB. Store at -20°C.