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.