[Histonet] cryoprotection help rat brain

From:N Fournier


I have been having some problems with our laboratory protocol for making up a viscuous solution for rat 
brain section.    We use the protocol of Watson and colleagues published in 1986 in Peptides (page 155-59). 

The original protocol states to make 1 L cryoprotectant solution one needs 500 ml of PB, 300 g sucrose (30% 
w/v), 10 g polyvinylpyrrolidone (1% w/v), and 300 ml of ethylene glycol (30% v/v).   I noticed that once I made 
up this solution and placed it in our -20 degree C freezer, the solution seemed to precipitate and become very 
viscous (strands of gelatinous material in the soluion).    The solution looked clear before I placed it in the 

Has anyone encountered this before?   Should I have let the solution stir overnight before placing in the fridge?  
Once the solution has been put in the fridge, taken out because of the observation above and restirred at room 
temperature, is it alright to return the solution bottle back to the fridge after that, or is the solution ruined.   Lastly, 
can I transfer brain sections into a cryoprotectant solution at -20 degrees if they have been cut recently and 
stored in PB at 4 degrees C?

I appreciate the help,


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