I have preserved large sections of sheep brain tissue in an ethylene glycol solution for later use. I'm hoping to take the sections out of solution and then slice on a microtome or cyrostat. How do I get the chunks of brain out of what appears to be squishy ice? Can I thaw slowing at 4 C ?
Hello Christine Windsor,
In order to use "preserved" tissue, you could also cut them using a microtome. However, you have to preserve them before (at Room Temperature) by fixing them in Bouin’s solution (HT10132; Sigma-Aldrich®) for IHC or formaldehyde solution (F8775; Sigma-Aldrich®) for IF during hours (or days, amoung the size of your tissue). Then, tissues have to be dehydrated in a graded series of ethanol and embedded in paraffin. Sections (often 3-5 µm thick) could be carried out using a microtome (Leica Microsystems© GmbH, for example).
If you have some squishy ice in ethylene glycol solution, you could warm up tissues at 4°C before to extract then fix them.
However, how do you (or another person in the Lab) "preserves" this tissue (EG alone? with an unconstolled slow freezing procedure at -80°C)? Indeed, there are as many freezing (and thawing) techniques as there are tissue types and the quality of the tissue (and IHC or IF) will be dependant.
The mains criteria to cryopreserve tissue are:
• The cryopreservation step with the choice of serum and penetrating (e.g., DMSO, Propylene Glycol, Ethylene Glycol, etc...) and/or external (e.g., sucrose, raffinose, etc...) cryoprotective agents
• The evaluation of the soaking temperature to break the supercooling point (with the use of seeding for example)
• The Thawing step
• The survival rate of tissue (and cells)
Hoping that this comment will be useful. Feel free to discuss or share some informations about your technical study.
Ludovic Dumont, Ph.D.
Hallo Christine,
are your sheep brain slices fixed in 4% PFA or formaldehyde before you store them in ethanol glycol? If this is the case then you have to remove the ethanol glycol from the tissue because it prevents freezing when you like to cut the tissue on a cryostat. I recommend to rinse the tissue in mixture out of ethanol glycol and 30%sucrose in PBS à/à; increase the concentration of sucrose step by step until pure 30% sucrose . Now you have cryoprotected your sections and can start cuttting it on a cryostat or freezing mycrotome. If you have to fixate your tissue at first, you have to exchange the ethanol glycol to 4% PFA, formalin or any other kind of fixation medium. Do it in the same I discribed it for the cryoprotection. Do it in the fridge. After the fixation step add the cryoprotection in 30% sucrose. Now you can star with the sectioning.Afterwards you can add any histological staining or immunohistochemistry. Good luck!
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