Paracelsus Medical University Salzburg
Question
Asked 30th Oct, 2015
What is the difference between fresh frozen tissues and tissues frozen in tissue freezing medium?
Hello everyone,
I have some mouse tumors that I am going to harvest. The vet who performs the surgery will place them in an isotonic saline solution, which they will be in for a brief time (< 1 hour) before I can get them back to the lab. I have read many protocols but I still don't know which is best. Right now I see my main assays being H&E, so what I care most about is preserving the morphology and tissue structure. How long could they be left in the saline solution in the fridge at 4 °C? I assume though that they would be better frozen as soon as possible. I will embed them in tissue freezing medium for cryosectioning. Is it better to store tissues by themselves in -80 °C or first embed them in tissue freezing medium. How important is the freezing rate depending on whether the tissue is embedded in tissue freezing medium or not? The cryomicrotome system that I use has a small area on it that gets down to around -60 °C. If I place the specimen in a plastic mold or a metal mold and freeze it in that small area, is that fast enough? What about just placing it in the -80 °C freezer to freeze? Finally, are snap-freezing protocols or sucrose cryoprotection protocols something I should look into in conjunction with tissue freezing medium embedding? Thank you.
Update (11/12/2015). The undergraduate student who performed the cryosectioning made the mistake of freezing some of the initial samples straight after removal from the saline solution at -80 C. We examined both mouse tumor and mouse spleen. For the pre-frozen samples, we decided to do fixation followed by cryoprotection in 15% and 30% sucrose on those samples anyways, finally embedding in OCT compound. The spleen had many tears, especially at 10 micron thickness, specifically near the center. A 20 micron thickness gave us the best results, with much less tearing and even some whole slices. The tumors appeared to hold up better despite the initial freezing compared to the spleens. 10 microns did give us more smaller tears running through the tumors, and 20 microns again gave us the best.
With the fresh samples, they cut great, but it seemed that they started to crack a short time after they were adhered to the slide. Is there anything we can do to avoid this?
Most recent answer
Apologize for lengthiness!
Good evening!
Sebastian:
I wonder at the issue: "initial storage of the material <by the vet who performs surgery> in isotonic saline solution, which they will be in for a brief time (< 1 hour)"
I bet you have to solve that issue before you think about freezing or not freezing (as you said: <my main assays being H&E, so what I care most about is preserving the morphology and tissue structure>.)
It might be that isotonic saline "is good enough for H&E" (as usually is anticipated from people knowing not much about histology) and "maintaining/preserving the morphology and tissue structure"..... also the type of specimen / tissue/ organ was not mentioned... but (un-)fortunately having been in the "real classroom and learned my lessons regarding tissue morphology and its preservation" over the last 34-35 years
I am convinced that storage of tissue from human or animal (depending not only on the tissue/organ type but also on volume/size and the quality of the surgical procedure) in physiological saline (which per definitionem should be "0.9% NaCl solution") might compromise as early as 5-10 minutes at RT (room temperature) the morphology (and probably also the properties of tissue for further experiments like Immuno and or retrieval of NAc's) to a certain and sometimes also visible effect in / by LM.
I can tell you this having seen the effects of storage of muscle samples for 15 mins at the maximum before (chemical) fixation (in comparison to muscle tissue IMMEDIATELY fixed after excision from the in situ area in the surgical theatre), especially in semithin (= 1µm resin embedded) sections.
For me only it would be o.k. to "clean/wash" shortly the (perhaps bloody) excised tissue IN/with physiological saline (but could be omitted completely because of a washing and), then following storage in e. g. (really physiological) phosphate buffer (composition = pH and tonicity will depend on the type of tissue material). Another thinkable possibility would be a so called "transport medium" or "transport buffer" like e. g. RPMI etc.
Last but not least, if your main task is only histology, H&E and morphology /structural preservation,
as has been said already (by Thomas, Mohamed, Matthew, Natalie and Vivica) the fabrication of say
buffered 4% Formaldehyde-solution ( = comparable / similar to 10% NBF=neutral buffered 10% FORMALIN) handed over to the <vet> in proper (tightly lockable plastic) specimen vials and his depositing the freshly excised tissue immediately into the solution is recommended. Then you can process FFPE as usual.
Freezing of tissue at -25°C or even at lower degrees might compromise your tissue-properties (in any respect, parameters naturally again volume/size of tissue), state of the art would be (cf. also muscle specimen processing for cryosectioning) rapid freezing in isopentane at minimally -85°C, optimally between -140 to -149 °C (precooled by liquid nitrogen until/at the point of obvious solidification), provided previous "embedding" in e.g. O.C.T.-medium. Cf. article
<Tissue Triage and Freezing for Models of Skeletal Muscle Disease, MENG et al, 2014, J. Vis Exp. 2014; (89): 51586>, to be found via PMC (PubMed Central) with free access (at least to me) @. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215994/
Storage (short or long term) in frozen state (with proper shielding and under standardized conditions at least in a -80°C refrigerator/low temperature freezer).
Good luck and best wishes, Wolfgang
1 Recommendation
Popular answers (1)
University of Central Florida
If your main assay is histological analysis then do not freeze at all but fix in formalin. Frozen sections are rarely as good as sections from formalin fixed tissues when it comes to preservation of tissue morphology.
If you still want to freeze, put tissue first in tissue freezing medium, then freeze. A more detailed discussion on this topic can be found here on Researchgate, foe example:
There is no universal fixation/tissue preservation method. It depends on what your plans are, what you want to study, what downstream applications you plan to do.
Many antibodies for IHC work on formalin-fixed paraffin-embedded tissues (FFPE), many do not. you can get decent protein for Western blotting from FFPE sections but snap-frozen tissue is better. Same is true especially for DNA and RNA.
Not sure how well DNA and RNA preserve after PFA/formalin fixation and freezing. But if the ratio of tissue to fixative and the size of the tissue are in a certain range, fixation should be fast and nucleic acid degradation at an acceptable level.
5 Recommendations
All Answers (7)
University of Central Florida
If your main assay is histological analysis then do not freeze at all but fix in formalin. Frozen sections are rarely as good as sections from formalin fixed tissues when it comes to preservation of tissue morphology.
If you still want to freeze, put tissue first in tissue freezing medium, then freeze. A more detailed discussion on this topic can be found here on Researchgate, foe example:
There is no universal fixation/tissue preservation method. It depends on what your plans are, what you want to study, what downstream applications you plan to do.
Many antibodies for IHC work on formalin-fixed paraffin-embedded tissues (FFPE), many do not. you can get decent protein for Western blotting from FFPE sections but snap-frozen tissue is better. Same is true especially for DNA and RNA.
Not sure how well DNA and RNA preserve after PFA/formalin fixation and freezing. But if the ratio of tissue to fixative and the size of the tissue are in a certain range, fixation should be fast and nucleic acid degradation at an acceptable level.
5 Recommendations
I agree with Thomas
my protocol is fixation in 10 NBF (3 hrs to overnight) then cryoprotect them in sucrose 15% then 30% till sinking, then put in OCT compound and store at -80C
You should put your frozen samples inside the cryostat at least 20 min before sectioning to be adapted to the new temperature
1 Recommendation
Icahn School of Medicine at Mount Sinai
Fixation in formalin and paraffin embedding will give you good morphology. Depending on your IHC protocol you might want frozen tissue for your antibody. If your material is in saline rather than flash frozen in liquid nitrogen, then you might want a 4 % PF, cryoprotectant, and sucrose solution before embedding in OCT and then freezing at -80oC. You should compare both frozen and fixed tissue, as the results may vary. Good luck!
1 Recommendation
Universität zu Lübeck
For pathology/morphology I would agree with the others and use formalin without any salin steps.
For IHC I would ask the veterinarian, if it is possible that he embeds the tissue in some O.C.T. medium in a metal mold and place it on dry ice if you don't have liquid nitrogen. Once it it frozen never thaw it again and store it at -80°C or colder until cutting it.
Paracelsus Medical University Salzburg
Apologize for lengthiness!
Good evening!
Sebastian:
I wonder at the issue: "initial storage of the material <by the vet who performs surgery> in isotonic saline solution, which they will be in for a brief time (< 1 hour)"
I bet you have to solve that issue before you think about freezing or not freezing (as you said: <my main assays being H&E, so what I care most about is preserving the morphology and tissue structure>.)
It might be that isotonic saline "is good enough for H&E" (as usually is anticipated from people knowing not much about histology) and "maintaining/preserving the morphology and tissue structure"..... also the type of specimen / tissue/ organ was not mentioned... but (un-)fortunately having been in the "real classroom and learned my lessons regarding tissue morphology and its preservation" over the last 34-35 years
I am convinced that storage of tissue from human or animal (depending not only on the tissue/organ type but also on volume/size and the quality of the surgical procedure) in physiological saline (which per definitionem should be "0.9% NaCl solution") might compromise as early as 5-10 minutes at RT (room temperature) the morphology (and probably also the properties of tissue for further experiments like Immuno and or retrieval of NAc's) to a certain and sometimes also visible effect in / by LM.
I can tell you this having seen the effects of storage of muscle samples for 15 mins at the maximum before (chemical) fixation (in comparison to muscle tissue IMMEDIATELY fixed after excision from the in situ area in the surgical theatre), especially in semithin (= 1µm resin embedded) sections.
For me only it would be o.k. to "clean/wash" shortly the (perhaps bloody) excised tissue IN/with physiological saline (but could be omitted completely because of a washing and), then following storage in e. g. (really physiological) phosphate buffer (composition = pH and tonicity will depend on the type of tissue material). Another thinkable possibility would be a so called "transport medium" or "transport buffer" like e. g. RPMI etc.
Last but not least, if your main task is only histology, H&E and morphology /structural preservation,
as has been said already (by Thomas, Mohamed, Matthew, Natalie and Vivica) the fabrication of say
buffered 4% Formaldehyde-solution ( = comparable / similar to 10% NBF=neutral buffered 10% FORMALIN) handed over to the <vet> in proper (tightly lockable plastic) specimen vials and his depositing the freshly excised tissue immediately into the solution is recommended. Then you can process FFPE as usual.
Freezing of tissue at -25°C or even at lower degrees might compromise your tissue-properties (in any respect, parameters naturally again volume/size of tissue), state of the art would be (cf. also muscle specimen processing for cryosectioning) rapid freezing in isopentane at minimally -85°C, optimally between -140 to -149 °C (precooled by liquid nitrogen until/at the point of obvious solidification), provided previous "embedding" in e.g. O.C.T.-medium. Cf. article
<Tissue Triage and Freezing for Models of Skeletal Muscle Disease, MENG et al, 2014, J. Vis Exp. 2014; (89): 51586>, to be found via PMC (PubMed Central) with free access (at least to me) @. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215994/
Storage (short or long term) in frozen state (with proper shielding and under standardized conditions at least in a -80°C refrigerator/low temperature freezer).
Good luck and best wishes, Wolfgang
1 Recommendation
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