[Histonet] Rat Brain/Cryosectioning

From:Neil Fournier

Dear colleagues,

Does any one have experience using liquid nitrogen cooled isopentane (2-methylbutane) on fixed (4% paraformaldehyde) rat brain tissue to quick freeze prior to sectioning (at approx 40 to 50 micron thick) on a cryostat?  I would like to know if I am doing the procedure correctly.   Our procedure is to block the brains and place them cryomolds that we then fill with OCT.  We then place a small container of isopentane in the liquid nitrogen, and then submerge the entire cryomold block in cooled isopentane.  I was told from colleague not to submerge the entire block in the solution, but I can't see any particular reason for not doing so.  Lastly,  how long should one typically keep the tissue submerged in the cooled isopentane?  And how long should one keep the isopentane in the liquid nitrogen before using for freezing the biological tissue?  

Also does any one have strong reservations against using the liquid nitrogen cooled isopentane method for freezing rat brain tissue prior to sectioning?  I have read that some use dry ice cooled isopentane; however, I have not seen any adequate explanation for one method over the other.

Thanks for the help,

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