RE: [Histonet] Freezing tissues
Isopentane will freeze before it reaches the temperature of liquid nitrogen. I often freeze tissues with liquid nitrogen, in fact it is my preferred method. Pour the nitrogen into a suitable container. I use a shallow styrofoam box, the lower portion of an ordinary styrofoam shipping container that I cut down to size. But just about any container will do, plastic or pyrex glass. Wait a minute until the boiling action stops. Put your specimen and OCT in the mold as usual, but make the mold so that it extends well above the surface of the OCT. I usually use polyethylene molds but I also use foil sometimes, for specimens that don't fit well into the polyethylene molds. Float the mold on top of the nitrogen. If it won't float without tipping over, hold it in position with forceps. When it is almost frozen, having just a small window of unfrozen OCT on the top about 1/3 the width of the block, remove it and transfer directly into the cryostat or the -80 freezer. The rest of the OCT will quickly freeze from the frozen OCT underneath. Don't let the block sink in the nitrogen, and don't leave it longer than I described above or the OCT may crack.
> From: firstname.lastname@example.org on behalf of Martin S.
> Sent: Thursday, January 4, 2007 2:08 AM
> To: email@example.com
> Subject: [Histonet] Freezing tissues
> Hi there,
> I need to freeze some mouse spleens tomorrow for cryosectioning. Usually
> I cut the spleen in half and place it in OCT in a foil mould. I then
> place the foil mold in a petri dish of isopentane sitting on dry ice.
> This seems to work well and I dont have problems with ice crystals or
> freezing artifacts. However, after the holidays there is not a drop of
> dry ice to be found in our institute so I need some advice on another
> method - possibly using liquid nitrogen. I've read a number of methods
> whereby you immerse the tissue directly in the LN, hold the tissue in
> the LN vapours or do as I do with isopentane but have the petri dish
> floating on LN (is this possible?).
> Any suggestions greatly appreciated!
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