I have tissue that has been frozen in N2 without supporting medium (OCT). Could I put the tissue in OCT and put next in isopentane just to solidify the block and make the sectioning possible.
Hello Anny
I routinely section frozen tissue without embedding in OCT, in fact I prefer to do it this way. I find that one has to optimise the cutting temperature to suit the tissue, and this temperature is usually a couple of degrees warmer than you would use with OCT-embedded material. I use a small amount of OCT to "glue" the tissue to a small cork disk which is itself frozen onto the cryostat chuck. This prevents the freeze/thawing effect described previously.
Your tissue may struggle with ice crystal artefact due to growth of ice crystals during freezing in N2, but the extent will depend on the tissue type.
Hi!, Anny,
It looks very hard to make a beautiful specimen, because the standard process is designed to prevent the tissue damage by ice. Without fixation, the tissue can be damaged, which is making pores and physalis in the cells. The other risk is Thawing and Refrozen process, which is also damaging it.
Hello Anny
I routinely section frozen tissue without embedding in OCT, in fact I prefer to do it this way. I find that one has to optimise the cutting temperature to suit the tissue, and this temperature is usually a couple of degrees warmer than you would use with OCT-embedded material. I use a small amount of OCT to "glue" the tissue to a small cork disk which is itself frozen onto the cryostat chuck. This prevents the freeze/thawing effect described previously.
Your tissue may struggle with ice crystal artefact due to growth of ice crystals during freezing in N2, but the extent will depend on the tissue type.
Your suggestion is very helpful. I have N2 frozen cardiac tissue (left ventricle). We will tray it immediately and share the result. Thank you gain.
I do agree with Kevin. Once your tissue is frozen there is no need to make a block with OCT. You can place it directly in the criostat holder with a drop of OCT or PBS.
At a molecular level, freezing affects membrane lipids, proteins and nucleic acids by changing the hydrophobic and hydrophilic interactions determining structure and function. This in turn adversly affects the ability of primary antibody binding and recognition of antigenic sites. Also without cryoprotection, tissue will show freeze fractures making structural identity very difficult. Unless compared to correctly processed tissue, the results yielded will never be 'true' in so far as correct preservation of structural components are concerned.
- Badges
- Science method