I'd like to flash freeze PFA-fixed mouse brains in OCT. Usually, this is done by flash freezing with liquid nitrogen, or dry ice. I was just wondering if it would be enough to transfer the samples in OCT straight to a -80oC freezer instead? I'd like to avoid any ice crystal formation.
You can gradually change the temperature,Initially move the tissue to PFA with 4°C then to ice chilled PFA and finally to -80°C. How ever I am not sure about crystal formation until the tissue is dry because the PFA also contains water percentage in it. If you change the temperatures without using PFA may allow the tissue to absorb moisture and sudden change in temperature may cause changes in tissue architecture which may affect the sectioning reporting. Get more suggestions form any expert pathologist.
It's better to cooling gradually with isopentane and liquid nitrogen. It prevents the formation of crystals.
But, more important. You can add steps of cryoprotection with glucose after freezing. You can put the samples into 10% glucose in PBS, 20% glucose in PBS and 30 % glucose in PBS. The time can be variable (at least 2 hours in my samples). The sample will float at first. To assure the cryoprotection, the sample should go down in the solution. After that, you can freeze the samples.
I hope it works for you.
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