Re: [Histonet] Percent Sucrose for cryopreservation

From:John Kiernan

According to an EM and X-ray diffraction stuudy (J.Lepault et al 1997; J. Microsc. 187: 158-166) you need 60% sucrose to prevent ice crystal formation. Most people use 20-30% sucrose, and combined with rapid freezing this is OK for light microscopy. I guess the ice crystals are too small to make visible holes in the tissue. Is there any point dissolving the sucrose in phosphate buffer rather than water? 

----- Original Message -----
From: "Pixley, Sarah (pixleysk)" 
Date: Monday, May 19, 2008 17:37
Subject: [Histonet] Percent Sucrose for cryopreservation

>  Dear List:
> What are the considerations in choosing the percent of sucrose for
> cryopreservation for frozen sectioning? Here is what we are doing:
> We perfuse mice with 4% peraformaldehyde in 0.1M phosphate 
> buffer, then
> fix overnight in the same. We dissect out either brain tissue or nose
> tissue. The nasal tissues are decalcified with 10% formic acid, then
> rinsed. All tissues are then immersed currently in 30% sucrose 
> in 0.1M
> phosphate buffer. We freeze in OCT compound using dry ice. Cryostat
> sections are cut at 14-20 um, depending on the tissue. We then do
> immunostaining for a variety of antigens, including BrdU. 
> Our question is: Is 30% sucrose optimal or should we use a lower
> percentage? We have heard that 30% may be too high. How would we
> determine that?  We have not had any problems, so we are 
> inclined of
> course to let it go, but perhaps we could optimize it for this next
> important set of experiments. 
> Thanks,
> Sarah Pixley
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