I am wondering why my section cracking sometimes happened. But actually I use the same protocol everytime as shown below. But the result is unstable. Please help to address. THanks a lot!
My Protocol:
1. Put the brain into 4% PFA for 24 hours.
2. Transfer the brain into 30% sucrose and keep for 2 days.
3. Take out the brain and dry it completely before putting the OCT.
4. Store the block at -80oC overnight.
5. Perform cryosectioning at -25oC
Dear Louis,
You don't mention where the cracks are in your tissue. If it is cracking out of the surrounding OCT, you may still have excess sucrose surrounding the tissue. I always put a big blob of OCT on my tissue and mix it around in it with my forceps until the Schlieren lines disappear, and then put the tissue into the mold with fresh OCT. Make sure to get the bubbles out also.
I prefer to freeze my blocks in molds (as well as fresh brain tissue) on powdered dry ice, since I get less cracking of my blocks that way. I have to be much more careful when using liquid N2 so that I don't get cracks in my block during the freezing process. I do not recommend trying to freeze blocks by just placing them in the -80 freezer since they don't freeze evenly(air is a poor medium for heat transfer). Since I am in Southern California, I also have to be careful that the blocks don't dry out during storage, so I also make sure to store them in a plastic box inside of a freezer-weight Ziplock bag.
If the tissue is cracking internally, it could be that your block is too cold when you section. Make sure to let it equilibrate in the cryostat to the cryostat's temperature before cutting. Trying to cut a block straight from -80 will usually result in cracks.
You don't mention what thickness of sections you cut, but you might also try cutting at a warmer temperature if you want 10-15 um sections. There are days that I have to tweak the cryostat temperature by a degree or two to get good sections.
Another possibility is that the brain is not fixing well with immersion fixation. I prefer to perfusion fix brain tissue, since it results in much better preservation of structures, and my primary antibodies work fine on tissue fixed int this manner. If that is not possible, make sure to use at least a 10:1 ratio of fix to tissue. A ratio of 20:1 is even better.
Also check the sharpness of the knife and the knife angle.
Lastly, open bottles of OCT don't last forever- it dries out a bit and becomes kind of rubbery. If it has become difficult to cut, it may be that it is too old (in fact, that is usually my clue that it is time to open a new bottle). Save the old bottle for attaching the block to the chuck, and switch to a new bottle for embedding tissue.
Good Luck!
Jill
Dear Louis,
You don't mention where the cracks are in your tissue. If it is cracking out of the surrounding OCT, you may still have excess sucrose surrounding the tissue. I always put a big blob of OCT on my tissue and mix it around in it with my forceps until the Schlieren lines disappear, and then put the tissue into the mold with fresh OCT. Make sure to get the bubbles out also.
I prefer to freeze my blocks in molds (as well as fresh brain tissue) on powdered dry ice, since I get less cracking of my blocks that way. I have to be much more careful when using liquid N2 so that I don't get cracks in my block during the freezing process. I do not recommend trying to freeze blocks by just placing them in the -80 freezer since they don't freeze evenly(air is a poor medium for heat transfer). Since I am in Southern California, I also have to be careful that the blocks don't dry out during storage, so I also make sure to store them in a plastic box inside of a freezer-weight Ziplock bag.
If the tissue is cracking internally, it could be that your block is too cold when you section. Make sure to let it equilibrate in the cryostat to the cryostat's temperature before cutting. Trying to cut a block straight from -80 will usually result in cracks.
You don't mention what thickness of sections you cut, but you might also try cutting at a warmer temperature if you want 10-15 um sections. There are days that I have to tweak the cryostat temperature by a degree or two to get good sections.
Another possibility is that the brain is not fixing well with immersion fixation. I prefer to perfusion fix brain tissue, since it results in much better preservation of structures, and my primary antibodies work fine on tissue fixed int this manner. If that is not possible, make sure to use at least a 10:1 ratio of fix to tissue. A ratio of 20:1 is even better.
Also check the sharpness of the knife and the knife angle.
Lastly, open bottles of OCT don't last forever- it dries out a bit and becomes kind of rubbery. If it has become difficult to cut, it may be that it is too old (in fact, that is usually my clue that it is time to open a new bottle). Save the old bottle for attaching the block to the chuck, and switch to a new bottle for embedding tissue.
Good Luck!
Jill
Dear Louis,
I just found this excellent publication, complete with images, posted by another member of RG in response to a similar question. I hope it helps:
https://www.feinberg.northwestern.edu/research/docs/cores/mhpl/tissuefreezing.pdf
Best,
Jill
Please see this link, it is useful for you:
https://www.researchgate.net/post/How_to_avoid_cracking_of_tissue_during_cryosectioning_of_human_formalin_fixed_brain_embedded_in_OCT
Hello
Kindly look at links
https://www.researchgate.net/post/Please_anyone_give_me_some_advise_how_to_prevent_cracking_of_rat_brain_slices_during_microtome_sectioning
https://www.feinberg.northwestern.edu/research/docs/cores/mhpl/tissuefreezin
Good Luck
There is a difference from my experiment method. 1. Extract the brain and freeze it directly to liquid nitrogen. 2. Transfer from liquid nitrogen to deep freezer at -80°C. 3. After 20 minutes, put the sample in O.C.T. Compound and freeze it in -80°C deep freezer. 4. Cryosection at -20°C
Brain Tissue cracking issues are:
(1)Grid-like impression artefact in small biopsies after using sponge pads
(2)To hold biopsies during using calcium neutralise formula.
(3)To obtain a section from tissues which are hard to cut after conventional fixation and processing.
(4)fragmented ,crushed and overstained sections from small biopsies .
(5)Find a minute biopsy after processing.
(6)Shattering,cracked folded sections from large blocks processed through paraffin wax.
(7)Recovering tissue that has dried out after malfunction of a tissue processor.
(8)To obtain a section from tissue which are often brittle after.
(9)Crooked or uneven ribbons
(10)Wrinkles appearing on sections floated on a float bath.
(11)Cutting thick and sections
(12)Wrinkles appearing in sections float in the floatation bath.
(13)Sections adhering to the block on the unstroke of microtome.
(14)Ribbons split vertically or scratch lines appearing in a sections.
(15)Sections crumble or tear
16)Cutting thick and thin within a minute high pitch vibration in a knife edge (chatter).
(17)Sections will not ribbon
(18)Sections roll up when cut.
(19)Sections disintegrate in the water bath.
(20)Sections appear chalky white on floatations
(21)To restore good nuclear staining when using hand staining or an automatic staining .
22) sudden or unusual stain automatic machine .
(23)How to restore good nuclear staining to tissue sections which have been exposed to over fixation or over decalcification.
(24)To distortion on slide to be stained by another techniques.
(25)lnadequate staining and /or leeching of eosin from tissue sections after washing and before dehydrating.
(26)A diffuse staining all structures with schiff reagent following fixation of tissue with schiff reagent following fixation of tissue with glutaraldehyde contacting fixatives.
(27)Muddy background staining when using verhoeffs elastic stain with a van gieson counter stain.
(28)Staining of cryststalloid mucoid material in the surface of a sections or smears where the tissue has a high muscin content.
(29)poor staining or lack of staining with Alcian blue dissolved in a acetic acid.
(30)To remove a cover glass from a slide more than 20 years old .
(31)Clearing hardened paraffin wax of benches and embedded centres
(32)Cells in a stained tissue sections appearing crenated.
(33)To recover sections from broken slides
(34)plastic coverslip tape lifting from a glass slide.
Jill Miotke
Thanks for your detail explanation. I was successfully finish the sectioning before following your instruction.
However, now I am doing 10-um sectioning for Nissl staining for cell quantification:
My protocol is below:
1. Mice are perfused with 4% PFA
2. Post-fixation of 4% PFA for 24 hours
3. Replaced with 30% sucrose (made with 0.1M phosphate buffer pH 7.4 and make up with MiliQ)
4. Keep in 30% sucrose for 2 weeks (replaced with new 30% sucrose in between for once)
3. before doing sectioning, make the block with OCT, and then put the block on ice bath for 10min, and then transfer it onto dry ice block for 15min (let it to be freezed slowly)
4. After that, put into the cryostat
5. Cut the section in the crytostat under -25oC
How can I improve it?
Is it necessary to keep the sample in 30% sucrose for 2 weeks? I think osmosis should be finished within a week , right?
Want to understand the problem in a scientific way.
anyone can help please. appreciate that.
Thanks
Louis
Dear Louis- I keep my mouse brains in sucrose until they sink to the bottom of the container, which usually means that the tissue is sufficiently protected. The adult mouse brains that I section usually sink in 48 hours at 4 C. You can speed this process up by placing the containers on a rotator if you have one that is safe for use in a cold room.
Good Luck!
Jill
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