I acquired frozen skin biopsies at -20 C degree from the hospital, and transferred them to the lab and stored at -80 C. (Because the tissues need to be stored for a few months, I did not store them at -20 C)
Before cryostat sectioning, I put the frozen blocks in cryotome for 1h so they are warmed up to -20 C. However, I still found freeze artifact on all my sections.
I was wondering if there is a better way to prevent this. Thanks!
I agree with Mohamed's protocol (NBF, sucrose, embedding), but we freeze the embedded tissue blocks in liquid nitrogen vapour before storing at -20 or -80 C.
But if you have months for sectioning, you should choose paraffin-embedding method; it results in better morphology than cryosectioning.
Honestly:
Dear Xu,
As long as "axiomatic" facts, physiological and physical necessities, and properties of "freezing" biological tissues (especially when dealing with specimens initially already frozen improperly) are ignored you might end up with the known phenomenon:
"garbage in, garbage out...." this was true in the 1970ies (when I started my studies) and is true still nowadays.
As you wrote:
"... acquired frozen skin biopsies at -20 C degree from the hospital, and transferred them....and stored at - 80°C..." [when I started my professional life in the EM-Lab I worked, I refused to take over specimens initially processed like "have been frozen at -20°C) when I was urged to process further for "scientifical analysis" or reasons...
You shouldn't be surprised about poor "morphology" or "artifactual structure of cryosections" ['freeze artifact on all my sections.' ] since the artifacts most obviously were produced already by the "freezing" procedure used in the hospital (slow freezing only to -20°C induces huge tissular alterations by formation of ice crystals, destroying cellular integrity). Then you put the specimen (poor freezing quality) into -80°C (perhaps you think this would save the integrity of your tissue piece [what were the initial dimensions?Was / Were the specimens protected from drying as this happens in a "fridge" by adsorption / condensation of water molecules over a certain period ]?) .
The criteria (to mentioned at least 3 I remember rapidly) to be met would be:
- size of tissue to be frozen must not exceed certain dimensions (the smaller the better)
- freezing (cooling) rate (depending on 'coolant' used, freezing capacity as mass/t)
- therefore a most rapid transition / transgression temperature lower than (minus) -80°C is mandatory.
Chemical prefixation (generally aldehydes, like PFA or FA) followed by cryoprotection (soaking the tissue after fixation in increasing %% sucrose solutions prior to freezing) not always is wanted or constructive (but may help a bit).
So - as has been intended to offer solutions to you by the previous posts of Mohamed and Nandor - physically (and perhaps also in terms of an expected achievement of "morphological equivalent" in your (stained) crysections - these leads (at least to me) seem to be rather along general lines....(especially handling of the specimen from the beginning on).
For your task (most probably) I would recommend to search for specific literature dealing with "freezing methods used for examination of biological (or even diagnostic human) tissue", especially the use of ==> "almost" solid isopentane *) precooled with LN2(liquid nitrogen) to a temperature for sure lower than (minus)-80°C [there are STANDARD/ROUTINE recipes and methods for freezing muscle biopsies state-of-art!],
as well as including the steps following, i.e., careful wrapping and further (also long time) storage minimum at -80°C (which is the recrystallisation point, i.e., growth of destroying ice crystals when temperature increases from -80° to e.g. -60°C...
*) = syn.also: 2-Methylbutan. "Isopentane is commonly used in conjunction with liquid nitrogen to achieve a liquid bath temperature of −160 °C" (for convenience only, quote from: https://en.wikipedia.org/wiki/Isopentane )
Best wishes and good luck !
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