Recently we are desperate about ice-injury of mouse brain slice in HE staining. There are so many small holes in our 8um slice, probably caused by ice-formation. We perfused our mice and used 20%(mass ratio, solute/solvent) sucrose to dehydrate their brains, still a lot of holes as a result. At freezing step, dehydrated brains were transferred immediately into -80 centigrade fridge, maybe it was not quick enough?
But some published paper also display photo with similar injury, so can this be fully avoided and how to mitigate as much as possible?
Thanks in advance for any advice!
you MUST freeze the brain over liquid nitrogen prior to placing at -80C. The slower you freeze the bigger the ice crystals grow, so they will bust your cells leaving holes! Also never store them at -20C.. you'll also ruin your tissue. You must freeze as rapidly as possible (snap freeze.) So, After your 20% sucrose step, place the brain for an hour or so in 20% sucrose and embedding media (like OCT) at 4C, then from there to neat (100%) embedding media in the final cryo-mold you want to store your tissue in. Leave this for an hour or so at 4C. Then, over liquid nitrogen, place a metal container of isopentane deep enough to submerge your brain/mold. make sure the metal container with isopent. is semi submerged in the liquid nitrogen, so that the nitrogen comes up almost to the top of your metal container with the isopent in it. Allow the isopent to get very cold,...when you see it turn white and thick at the bottom of the container then it is ready. gently place your brain mold into the freezing isopentane and allow it to sit for a while untill you no longer see any clear embedding media (it will turn opaque white). then leave it some more (it will take less than a minute in total for a rat brain, and about 20sec for a mouse brain). Use forceps to remove the mold from the cold iso. then immediately transfer the mold to dry ice or the -80C freezer. never let it thaw! ..hope this helps! good luck and happy lunar new year to you
Two things. 1) I'd recommend using 30% sucrose and 2) how is the freezing done?
Hi Hao Chen.
In our lab we usually cryoprotect (rat) brains after perfusion by leaving them over night in 30% sucrose (mass ratio) in PBS. They should sink to the bottom of the tube. That´s a good indication that the brain is saturated with sucrose. Then we freeze them carefully in ice cold (-20°C - -40°C) 2-methylbutane (glass jar on dry ice). The fluid shouldn´t be colder than -40°C (thermometer!), because the brain could brake in two parts! Leave the brain long enough in the fluid (10 minutes?). It gets a white color. After that, you can transfer it (on dry ice) to your freezer. Make sure it doesn´t thaw.
Hope I could help. Please let me know, if it worked.
Greetings Marcia
We had holes caused by water cristallization a few years ago, because we left the brains for a shorter period in methylbutane. But yes these holes can be prevented completely!
We use 2.1M sucrose in buffer at the final step. Start with infitration ,low, medium and than 2.1M. The brain should sink to the bottom. If not, leave it longer in. The speed of freezing is not an issue.
Heinz
In addition to the cryoprotection in 30% sucrose, the optimum temperature at which I section is -20°C (after initially freezing to -40°C). Probably it will help to look into that as well.
First, it is important to use 30% sucrose (or 20% glycerol/2%DMSO) to reduce ice crystal formation; secondly the formation of ice crystals occurs only when the tissue is cooled to slowly or thawed too slowly. Immersion in cold isopentane (as suggested by Marcia) gives rapid cooling; rapid thawing is achieved by immersing in a large volume of the cryoprotectant, prior to exchange for paraffin embedding or freeze cutting. The 20% glycerol/2%DMSO is superior to 30% sucrose as a medium for freeze cutting (for discussion see Rosene et al., J Histochem. Cytochem. 34: 1301-1315, 1986).
we don't have any holes our mouse brains, and I think it's not a matter of freezing the brain quickly enough. What's the procedure you use when taking the brains out of the -80 freezer and cutting them? Usually, having holes in the slices is because the tissue went through a freeze-thaw cycle.
Hi Yun, we used cryosectioning and transfered our sample from -80 to approximately -20 cryostat chamber as quickly as possible. In this case, it might cost 15 to 20 sec to transfer. We thought it is short enough but really enough to prevent thawing? Actually I'm surprised that thawing (per se?) can hurt tissue as well but is it the refreezing process that can cause holes?
Thank you all again.
Suspend a beaker of Isopentane, partially submerged in liquid nitrogen and allow to cool until it begins to visibly solidify from the bottom up. Immerse tissue completely in the near-frozen isopentane using a long metal forceps for 30 seconds and transfer to the cryostat or freezer as quickly as possible. The storage temperature of the tissue is not critical once it is below -20 C. Transfer from freezer to cryostat must be rapid. Tissue is not damaged by moving from -80C to -20C. The only way that ice-crystals can form and damage can occur is if any portion of the tissue rises above freezing point (0 C) - so always use pre-cooled metal forceps etc to handle the tissue. I used this technique with fresh muscle biopsies for over a decade without fail. We didn't use sucrose solutions but I believe that they can give some added protection.
Kind regards,
Kieran
Kieran is right. Isopentane is necessary to avoid an abrupt (rapid) freezing of tissue. The cryostat temperature for brain is around -15 C. Avoid touching the frozen tissue by hand.
You may try to restore the damaged tissue by bringing it to room temperature and refreezing in isopentane in liquid nitrogen. For muscle is ok.
best regards,
Emilia
When water is frozen its volume increases; if the slices are not fully dehydrated, the ice might cause holes that persist when the tisue is thawed.
Brain tissue holds a lot of water. The sucrose is not dehydrating tissue but acting as a cryoprotectant, meaning it is minimising the formation of large ice crystals. Once cryoprotected it is not necessary to freeze in isopentane. You could try starting with 10% sucrose until the tissue sinks then going to 20%, then 30% until the tissue has sunk again. Then freeze in -80C and hold there until transferring to the cryostat for cutting.
You dont mention whether you fix the tissue first with 4% paraformaldehyde which is necessary if you are using the sucrose. Unfixed tissue needs to be frozen using the above mentioned technique with isopentane in liquid nitrogen. Any warming of this tissue will result in larger ice crystal formation, which occurrs anyway but it is the size of the crystals which do the damage so you need to minimise this by using the mentioned techniques.
Hi everyone, your information is helpful! According to the paper recommended by Alexander, mechanical disruption on tissue comes from those aggregated big ice crystals, just as mentioned by Mary. And mechanism behind this, indicated in the paper, is the recrystallization when freezing and thawing, which causes initial small crystals to form really big ones especially at temperature above -30 (proceed rapidly above -20 degree, Asahina et al., 1970. ). Thus -20 cryostat is still not that perfect?
I'm not sure we touched our sample when transferring from freezer, but samples were stored in thin plastic gloves all along and weren't taken out until start cutting. And yes we fixed them with PFA overnight after perfusion. We'll further explore with your kind advice.
Dumb question: did you do a DAPI stain on your slices?
When I looked at my first immunostaining of a rat brain slice that I had made myself, I was shocked by how many holes there were - must be damage from freezing, I thought as well. Then I did a DAPI staining: all smaller holes were nuclei, all bigger holes were blood vessels (neatly lined with nuclei of endothelial cells in cross section). No evident freeze-damage at all, and it later turned out that even sub-cellular morphology was maintained perfectly.
I used a protocol similar to yours, without isopentane and processed in a -20 cryotome, but I went up to 30% sucrose. Another problem might be your slice thickness, 8 µm seems very thin and might tear during drying. In many cryotomes, it is virtually impossible to cut intact slices below 10 µm, and even below 20-30 µm can be challenging on some setups.
Hi Sven, remind you blood vessels are lined with endothelial cells.
Hao,
You got all the best solution, practically good cryo-protection may obtain by soaking brain block tissue in 30% buffer sucrose solution over night.
Hey, can you upload a picture or something of your HE stained slices and let us see just how big the damage is? Freezing without proper dehydration would cause ice crystals to form, but it's the thawing process that forms the holes. Oh, and as a sort of followup on Sven's question. What kind of cryotome did you use to cut the slices? We use a Leica CM 3050S, and getting nice beautiful slices at less than 20um is still quite challenging for us.
Hi Yun, here is an objective len 20x photo, not with high resolution though. Thanks for reminding me.
About the crytome, I need to check when I return or ask others perhaps... We used Leica too but I felt not so challenging to slice below 10um with it. In fact, more skilled experimenter can decrease to even 3.5um with little effort, no kidding.
Happy Lunar New Year :)
you MUST freeze the brain over liquid nitrogen prior to placing at -80C. The slower you freeze the bigger the ice crystals grow, so they will bust your cells leaving holes! Also never store them at -20C.. you'll also ruin your tissue. You must freeze as rapidly as possible (snap freeze.) So, After your 20% sucrose step, place the brain for an hour or so in 20% sucrose and embedding media (like OCT) at 4C, then from there to neat (100%) embedding media in the final cryo-mold you want to store your tissue in. Leave this for an hour or so at 4C. Then, over liquid nitrogen, place a metal container of isopentane deep enough to submerge your brain/mold. make sure the metal container with isopent. is semi submerged in the liquid nitrogen, so that the nitrogen comes up almost to the top of your metal container with the isopent in it. Allow the isopent to get very cold,...when you see it turn white and thick at the bottom of the container then it is ready. gently place your brain mold into the freezing isopentane and allow it to sit for a while untill you no longer see any clear embedding media (it will turn opaque white). then leave it some more (it will take less than a minute in total for a rat brain, and about 20sec for a mouse brain). Use forceps to remove the mold from the cold iso. then immediately transfer the mold to dry ice or the -80C freezer. never let it thaw! ..hope this helps! good luck and happy lunar new year to you
We have used 30% aqueous sucrose which prevents the ice crystal formation if the tissue has been in the sucrose long enough (a few days usually for brain) it must sink or some weight put on it to keep it immersed. Freezing can be effected on a dry ice copper stage or the other methods mentioned above.
Hi Hao Chen,
I am also facing same problem. Could you help me giving in details especially the frozen system using n-hexane...
Thanks
I disagree that the small holes are blood vessels. They are the result of ice crystal formation. In freezing tissue two things can avoid them. Placing them into isopentane over liquid nitrogen for unfixed blocks. OR placing fixed tissue into sucrose until it sinks then freezing it.
I also think that many of the small holes are freezing artefact and not blood vessels. We had good experience with wrapping the mouse brain in aluminium foil and then put it in liquid nitrogen cooled isopentan.
Best regards, Fred
- Badges
- Science topic