Does anyone have protocol recommendations for freezing/storing human uterine tissues? I have seen some protocols that place tissues directly in LN2 and then transfer to -80 for long term storage. Others wash with PBS prior to snap freezing. We have previously used RNAlater for samples, but this protocol is not great for immunohistochemistry. Long term, we would like to do DNA/mutation analyses and immunohistochemistry with these samples.
Hello Brianna N. VanNoy,
For a 'long term use' (i.e., DNA analyses), the sample could directly put into LN2 (onto a cryotube) without any specific protocol ajustement.
However, in order to cryopreserve tissue for immunohistochemistry, you have to use cryoprotective agents with an unconstolled slow freezing procedure at -80°C and higher quantity of theses components for vitrification (directly in LN2). Indeed, there are as many freezing (and thawing) techniques as there are tissue types and the quality of the tissue (if used for IHC or IF) will be dependant.
The mains criteria to cryopreserve tissue (for immunohistochemistry) 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)
The washing steps with PBS (or medium) have to be done to eliminate cryoprotective agents or wash the tissue.
Hoping that this comment will be useful. Feel free to discuss or share some informations about your technical study.
Ludovic Dumont, Ph.D.
I would strongly recommend to put the sample directly in LN2, or alternatively in isopentane cooled by LN2. PBS in my opinion has a detrimental effect.
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