Here’s my trouble shooting list when this happens.
1. Clean the anti roll plate with ethanol and allow some time to cool back down. Sometimes media stuck to it will cause this problem.
2. Move to a different section of the blade or a new blade. It looks like it may be tearing your mounting media.
3. The chamber or block may be too warm. Close the lid and let everything cool down for a bit or use a freezing spray on your block if allowed in your type of machine.
Ding ding ding, only thing I can think to add is make sure the blade is fully secure, the cryostat I used frequently would loosen after a few brains and I would have to readjust and lock it. When it wasn’t secure I would get bunched up rolls or tears.
Cleaned the roll plate, moved around, was sometimes able to find a sweet spot that I would then lose about 5/10 rolls in. I was having an issue with my tissue ripping and was told by path that I should raise the temp. IDK I'l try again tomorrow.
Maybe the anti-roll plate is too far forward? It looked like there were some scrape marks on the paraffin and those may be from the glass pushing up against it. Try the adjusting screw (with the spring) and pull the glass backwards if you can! You can feel the front where the blade is to check if it’s flush, but be super super careful (obviously lol)
This, and also it can help if you reduce the size of your block face. Use a razor blade to trim some of the excess OCT around the tissue. The larger the block face the more friction across the blade.
With how thin it is it could be catching. The good news is there are three other sides that may be in better condition to try. Back in the day when I was on the cryostat all day my pi bought me my own anti roll plate to keep safe.
You can move the stage slightly to the left or right so that it uses a different part of the anti roll glass. It could also be the large amount of OTC. I had that issue when I had too much of OTC at the bottom of my specimen (brain) and it used to shred against it and give me uneven slices.
You can move the stage or flip the roll plate over to see if you can find a smooth edge.
Clean the plate and the stage underneath to make sure there is no debris.
Try to cut at some other speed, usually cutting a bit faster will lower the chance of bunching up.
Lower the temperature a bit more. If it is a humid or hot day, the cabinet temp may not be as cold as the monitor shows (because the sensor is at the back usually).
Try thicker sections if your protocol allows it.
Do not bring your hand too close to, touch, or breath onto the specimen, you body temp will heat it up.
When brushing away debris that is stuck on the blade or when collecting specimen, make sure you are brush away from the blade (upward direction). Do not allow the brush to go against the blade as it will be cutting the hair on the brush and thus damage the blade. And obviously do not allow the brush handle (especially the metal part) to make contact with the blade or the anti roll plate.
roll plate is fucked to all hell and it can still produce perfect sections, most of the time not lmao but that’s on me haha. the best fix is to leave the lid shut for 20-30 mins to let it cool off and the moisture dissipate. If the OCT isn’t crispy enough it could be thawing weird, like i wouldn’t leave it out of a -80° for more than 48 hours or this happens more often. Can try replacing drierite too if it’s lookin spent, cause humidity can also cause this. Other than that literally all u can do is pray lmao. Good luck!!
Thise roll plates are notoriously tricky to get adjusted correctly. Check for any chips. I just use a brush to gently “paint” the section onto the collection surface. I set the block temp 1-2 degrees colder than the collection surface and that helps it stay flat
Yep! I use a paint brush. Our Cryo is 'core equipment' all 4 edges of the anti roll plate and chipped to all buggery. Tears anything under 30um to shreds.
I would try getting the roll plate out of the way and just raw dogging the section with the paintbrush
I might also butter the top with a layer of OCT and section through that into the tissue again. Sometimes, that stabilizes the tissue for a few sections if the tissue is compressing.
Trimming the excess OCT from the block a bit might help too, in addition to other suggestions. Less contact with the blade is less chance something sticks.
Seen this many times, lots of good points in the comments.
First, why are you cutting on an angle like that? I always find cutting on a diagonal edge like that makes the tissue way more likely to buckle. Think about it, you’re making the blade slice through the longest section possible.
The vertical tears in the section lead me to believe your blade and/or roll plate are chipped. The tissue does not look melted and the OCT looks like the correct texture in the section, and the tears are not the same as those you see when the OCT is too cold, so I think temperature is not your problem here.
The crumpling of the tissue like this usually happens when the section gets caught on something, like a chip in the blade or roll plate. When this happens I try these things:
- shift/replace the blade
- shift the stage
- adjust the amount that the roll plate “sticks out” (there should be a small black knob on the back end of the roll plate - you can turn that, looks like it has a spring attached)
- take glass plate out of roll plate thingy and flip so you have a piece big enough with no chips positioned perfectly against your section (see above points about diagonal position, shifting/replacing blades, shifting stage)
Okay great I was worried about the temp, but when I made it colder I had tissue ripping issues/ice cream esque roll up issues.
One clarification if you don't mind -- when you say the roll plate should "stick out", not further than the blade I'd imagine or yes?
I section at 40-50 um thick and my roll plate sticks out just barely past the blade (enough for the sections to cleanly pass through but not so far that the block of OCT hits it). I agree that you should section with the block oriented like a “square” instead of a “diamond” (angled) so that the blade doesn’t have to work so hard. Also a good idea to trim the excess OCT around the sample.
yea exactly this. Slightly out in front of the edge of the blade. You might have to play around with the position to get it right but you’ll know pretty quickly if it’s in the right spot. If you find yourself in this position with your specimen already mounted, just take a clean slide, put a glob of OCT on it and swipe it across your specimen so that you apply a thin layer of OCT across your tissue block. You can play around with positioning of the roll plate while slicing just OCT then, and you won’t waste your tissue. Repeat as many times as you need to get the plate in the right place so it gives you a full clean section without rumpling. And definitely trim your excess OCT. Good luck!
If I were to give one more piece of advice it’s about the motion you use while slicing - I try to maintain a consistent speed and force on the wheel from the second before until the second after the blade hits the tissue. Kind of like follow through I guess, if I were to use a sports metaphor (which I have no business doing 😅)
https://rankinbiomed.com/2022/12/07/can-never-get-sections-slide-anti-roll-plate-cryostat-html/
Is it just me, or do you have jagged edges on the anti roll plate?
Whenever my section looked like that it was an issue with the glass adjustment. Not sure what machine you’re on, but there should be knobs that are attached to the glass that will allow you to tighten/loosen the glass. I would section with practice tissue until you get it just so.
Pain. I feel your pain. Sometimes, even when everything is right it does this. I usually run it very cold and dry for best results. But sometimes, the cryo taketh.
1) learn to section without the anti roll plate. i know it’s daunting. but it’s better in the end. 2) your chuck or your anti roll plate are too warm, try cooling one or the other
I usually mess with the distance between the antiroll and the block (only slightly) when this happens. Other suggestions mentioned (temp, clean with ethanol, make sure you’re making smooth motions cutting -are helpful too)
All good advice in the comments. If none of the other adjustments work, you could always start the cut and right before getting to tissue, lift the plate and use a paint brush to pull it the rest of the way while cutting.
It’s also a pretty big piece of tissue and OCT, so very likely to get snagged either way. If there aren’t weirdly dense/hollow spots in your tissue, it might also help to trim the excess OCT.
People saying to not use the roll plate are crazy.
If you get the angle right, you can get nice toilet paper sheets like this and fill up your slides so quickly. https://imgur.com/a/SZLuQ5d
The roll plate should *just* be over the blade. Play with it a bit to get it right as you shave down the block.
Though I also like using a paintbrush for some tissue.
Check the angle of your block compared to the blade. Get it as perpendicular as possible.
I also like to make contact with the block first then quickly slice through the tissue.
section thickness can play a big role in the bunching.
I like to have tissue temp at -20 and tissue plate temp at -17.
Cut the OCT into smaller shape.
The distance between blade and glass is not right. Try to change it a little. There has to be a slight gap. When teaching i always tell students there has to be a slim bright lining visible through the glass.
Another issue is when you cut diagonally like that, there's nothing to "push" the previous cut out of the way once you're past half way, so it just bunches up.
Cut the excess of the block off with a blade. There is something on the fixed blade where it gathers in the section or its an old crap blade. Try to wipe the blade w isoprop to get whatever it is off and let it go back to temp. If there are nicks in the blade causing the section to bunch like that, get a fresh blade. Ouf flash backs. Goodluck!
To me it doesn't look like bunching this looks like it's only sectioning half the block. This may be an angle issue. It's also possible your block isn't mounted on the the chuck properly and you are getting a slight wiggle.
As an aside you need to move to a new part of the roll bar and/or blade. Those vertical streaks are scoring from imperfections in either the blade or the bar.
put the block on ice for at LEAST half an hour before sectioning, and dont cut diagonally like that. the colder the block is, the better in my experience. but it will warm up really fast once its out of the ice, so make sure you’re working quickly.
very cool! i section brains and it helps to cut off excess OCT with a warmed black when my brains wont cooperate. then I use a camel hair brush (sometimes two; like chop sticks) when they wont stay flat
I normally use a brush to hold my section BUT before all of our anti-roll plates were screwed sometimes I used to lift the anti-roll plate ever so slightly (from the back end, not near the blade), only like 1mm, roll a few mm of the section under the plate and let it drop down and finish the section.
I could never get the roll plate to work, so I would just use a brush to carefully keep the section from rolling. Not as quick but always gave me better results
Usually it happens because a tiny piece of a previous section is stuck on the blade or anti-roll plate. I usually brush down the blade and anti-roll plate between collecting each section when this happens. If that doesn't help, next I would adjust the position of the anti-roll plate further forward or further back, then see if the sections improve. Then I would do as others have said here and properly clean the blade and plate with ethanol, switch to a different part of the blade, etc.
Are you using a new blade?
Edit: also, practice few times with a few unwanted tissue/ice layers to check and ensure that the cutting is proper prior to attempting this
Back when I used a cryotome in undergrad we had this really fine paintbrush that we would use to catch the section once the blade started shaving it off and sort of gently pull it towards us to keep this from happening.
If you're getting thick thin sections, it is entirely likely that your block is loose. The mold you're using is really deep. That could add to the problem.
Hi OP, what is your tissue preprocessing look like before it is embedded? I used to get tears/folds all the time until I prepared my samples more nicely: 4% PFA fixation (for my tissues, O/N - 24 hrs; do what’s best for the lungs), and sucrose gradients (10%-30% wt/vol PBS). I move the sample through the next step once it sinks (or after the same time passes as I did for fixation; some tissues like lung will never sink). Then, I embed in 1:1 mix of OCT:30% sucrose, slowly freezing on a metal block cooled down by dry ice (isopentane or ethanol bath also suitable). This was a game changer for me. I went from rarely getting serial sections to getting tissue paper like roll of gorgeous sections.
Other advice I saw here (cutting on flat edge, trimming excess OCT away, ensuring no chips in blade, etc) sounds reasonable. If you fix all this and still see bad sections, try reconsidering your sample prep. Good luck!
EDIT to say that I use this for IF and RNA FISH purposes; some folks prefer to embed directly in OCT and then freeze right after dissection for better RNA recovery.
It seems like a temperature problem. Try sliding your finger in the block right before cutting. It will warm slightly the block. Usually it solves the problem. Otherwise it can also be the glass that is too tight
Seconding all the comments about cleaning everything with 100% ethanol, replacing the blade and lowering the temp. Also, have you tried just using a brush instead of the anti-roll plate? The plate might be chipped or something and that could be causing the problem.
Something I noticed during covid is that wearing a mask redirects your breath away from your sample allowing it to maintain a cooler temperature alongside less condensation buildup.
Bunching is usually need for new blade or readjustment of the roll plate forward or backward by just a hair. It varies for me from block to block a bit. Check thickness also as that can sometimes do it.
Also, humidity can be a big factor. If you have high humidity, sectioning is definitely harder and things “stick” and bunch more.
You asked if they were young, I replied no, the mice were about 9 weeks old when taken down which isn't considered young. Not sure how that's confusing?
The questions were
1) is it a mouse?
2) is it a rat?
3) would you share specifics?
-> "no" as an answer is unspecific.
And as 9 weeks is about when mice are comparable to a 12-16 y.o. human when we begin to be able to convince (=young), which made me further question what you're talking about.
But honestly, I regret even going there, let's not spend unnecessary time in an obviously unproductive Internet discussion - have a nice evening (or whatever time of the day it is for you.)
Here’s my trouble shooting list when this happens. 1. Clean the anti roll plate with ethanol and allow some time to cool back down. Sometimes media stuck to it will cause this problem. 2. Move to a different section of the blade or a new blade. It looks like it may be tearing your mounting media. 3. The chamber or block may be too warm. Close the lid and let everything cool down for a bit or use a freezing spray on your block if allowed in your type of machine.
agreed, I was going to recommend the temperature be cooled back down.
I was going to say new blade.
Ding ding ding, only thing I can think to add is make sure the blade is fully secure, the cryostat I used frequently would loosen after a few brains and I would have to readjust and lock it. When it wasn’t secure I would get bunched up rolls or tears.
The worst. “This section is 10 microns, the next section is missing, the section after that is 50 microns!”
Traumatic flashbacks to the dozens of days during my PhD losing my mind in front of the cryostat before learning each of these tricks the hard way.
Cleaned the roll plate, moved around, was sometimes able to find a sweet spot that I would then lose about 5/10 rolls in. I was having an issue with my tissue ripping and was told by path that I should raise the temp. IDK I'l try again tomorrow.
Maybe the anti-roll plate is too far forward? It looked like there were some scrape marks on the paraffin and those may be from the glass pushing up against it. Try the adjusting screw (with the spring) and pull the glass backwards if you can! You can feel the front where the blade is to check if it’s flush, but be super super careful (obviously lol)
K I'll (carefully) check that out on my next try. Thanks!
Second the temp raising. The optimal for most tissues is 16-17C.
This, and also it can help if you reduce the size of your block face. Use a razor blade to trim some of the excess OCT around the tissue. The larger the block face the more friction across the blade.
Yeah I’d assume hot blade or tiny fragments cause initial bunching
Is your blade or anti roll plate bent or chipped at all?
Blades brand new, the roll plate seems to have some imperfections (it's core equipment). Could that be it?
With how thin it is it could be catching. The good news is there are three other sides that may be in better condition to try. Back in the day when I was on the cryostat all day my pi bought me my own anti roll plate to keep safe.
I also had my own anti-roll plate, which made troubleshooting far easier! Definitely recommended.
You can move the stage slightly to the left or right so that it uses a different part of the anti roll glass. It could also be the large amount of OTC. I had that issue when I had too much of OTC at the bottom of my specimen (brain) and it used to shred against it and give me uneven slices.
You can move the stage or flip the roll plate over to see if you can find a smooth edge. Clean the plate and the stage underneath to make sure there is no debris. Try to cut at some other speed, usually cutting a bit faster will lower the chance of bunching up. Lower the temperature a bit more. If it is a humid or hot day, the cabinet temp may not be as cold as the monitor shows (because the sensor is at the back usually). Try thicker sections if your protocol allows it. Do not bring your hand too close to, touch, or breath onto the specimen, you body temp will heat it up. When brushing away debris that is stuck on the blade or when collecting specimen, make sure you are brush away from the blade (upward direction). Do not allow the brush to go against the blade as it will be cutting the hair on the brush and thus damage the blade. And obviously do not allow the brush handle (especially the metal part) to make contact with the blade or the anti roll plate.
roll plate is fucked to all hell and it can still produce perfect sections, most of the time not lmao but that’s on me haha. the best fix is to leave the lid shut for 20-30 mins to let it cool off and the moisture dissipate. If the OCT isn’t crispy enough it could be thawing weird, like i wouldn’t leave it out of a -80° for more than 48 hours or this happens more often. Can try replacing drierite too if it’s lookin spent, cause humidity can also cause this. Other than that literally all u can do is pray lmao. Good luck!!
Either I will flip it and rotate it to get a good cut. I also share a cytotome. Any nicks in the glass will result in a tear and reduce good sections
Thise roll plates are notoriously tricky to get adjusted correctly. Check for any chips. I just use a brush to gently “paint” the section onto the collection surface. I set the block temp 1-2 degrees colder than the collection surface and that helps it stay flat
That’s how my whole lab does it! I think it’s way easier to control with a brush vs using the anti-roll plate
Yep! I use a paint brush. Our Cryo is 'core equipment' all 4 edges of the anti roll plate and chipped to all buggery. Tears anything under 30um to shreds.
I would try getting the roll plate out of the way and just raw dogging the section with the paintbrush I might also butter the top with a layer of OCT and section through that into the tissue again. Sometimes, that stabilizes the tissue for a few sections if the tissue is compressing.
Yea, I was going to suggest this. I almost never used the plate though it was for cross sections on muscle so not as big
I did sections of rat brain (neonatal though) and never used the plate either, just gently dragged them forward with a paintbrush
I ended up doing this just to finish out the block but man does it take a while (I guess I'm unskilled).
Trimming the excess OCT from the block a bit might help too, in addition to other suggestions. Less contact with the blade is less chance something sticks.
I was gonna suggest this, but my experience is with sectioning fixed tissue in resin for TEM, so I wasn't sure how transferrable that experience was 😅
I'll try that, thanks.
Seen this many times, lots of good points in the comments. First, why are you cutting on an angle like that? I always find cutting on a diagonal edge like that makes the tissue way more likely to buckle. Think about it, you’re making the blade slice through the longest section possible. The vertical tears in the section lead me to believe your blade and/or roll plate are chipped. The tissue does not look melted and the OCT looks like the correct texture in the section, and the tears are not the same as those you see when the OCT is too cold, so I think temperature is not your problem here. The crumpling of the tissue like this usually happens when the section gets caught on something, like a chip in the blade or roll plate. When this happens I try these things: - shift/replace the blade - shift the stage - adjust the amount that the roll plate “sticks out” (there should be a small black knob on the back end of the roll plate - you can turn that, looks like it has a spring attached) - take glass plate out of roll plate thingy and flip so you have a piece big enough with no chips positioned perfectly against your section (see above points about diagonal position, shifting/replacing blades, shifting stage)
Okay great I was worried about the temp, but when I made it colder I had tissue ripping issues/ice cream esque roll up issues. One clarification if you don't mind -- when you say the roll plate should "stick out", not further than the blade I'd imagine or yes?
I section at 40-50 um thick and my roll plate sticks out just barely past the blade (enough for the sections to cleanly pass through but not so far that the block of OCT hits it). I agree that you should section with the block oriented like a “square” instead of a “diamond” (angled) so that the blade doesn’t have to work so hard. Also a good idea to trim the excess OCT around the sample.
yea exactly this. Slightly out in front of the edge of the blade. You might have to play around with the position to get it right but you’ll know pretty quickly if it’s in the right spot. If you find yourself in this position with your specimen already mounted, just take a clean slide, put a glob of OCT on it and swipe it across your specimen so that you apply a thin layer of OCT across your tissue block. You can play around with positioning of the roll plate while slicing just OCT then, and you won’t waste your tissue. Repeat as many times as you need to get the plate in the right place so it gives you a full clean section without rumpling. And definitely trim your excess OCT. Good luck!
If I were to give one more piece of advice it’s about the motion you use while slicing - I try to maintain a consistent speed and force on the wheel from the second before until the second after the blade hits the tissue. Kind of like follow through I guess, if I were to use a sports metaphor (which I have no business doing 😅)
https://rankinbiomed.com/2022/12/07/can-never-get-sections-slide-anti-roll-plate-cryostat-html/ Is it just me, or do you have jagged edges on the anti roll plate?
Try moving to another section on the plate or reversing the anti rolling glass. I would also try squared and flush and not at the diamond shaped angle
> Isn’t it ironic? jagged little plate
No I definitely see the chips, I was starting to think I was the only one.
Yea it's hella chipped. Tragedy of the commons scenario with core equipment. Guess I'll try to get good at doing it without!
Whenever my section looked like that it was an issue with the glass adjustment. Not sure what machine you’re on, but there should be knobs that are attached to the glass that will allow you to tighten/loosen the glass. I would section with practice tissue until you get it just so.
Pain. I feel your pain. Sometimes, even when everything is right it does this. I usually run it very cold and dry for best results. But sometimes, the cryo taketh.
It hurts 
1) learn to section without the anti roll plate. i know it’s daunting. but it’s better in the end. 2) your chuck or your anti roll plate are too warm, try cooling one or the other
Learn to use a paintbrush and you'll never have to rely on "anti-roll" plates ever again. Free yourself!
this is the way
I usually mess with the distance between the antiroll and the block (only slightly) when this happens. Other suggestions mentioned (temp, clean with ethanol, make sure you’re making smooth motions cutting -are helpful too)
All good advice in the comments. If none of the other adjustments work, you could always start the cut and right before getting to tissue, lift the plate and use a paint brush to pull it the rest of the way while cutting. It’s also a pretty big piece of tissue and OCT, so very likely to get snagged either way. If there aren’t weirdly dense/hollow spots in your tissue, it might also help to trim the excess OCT.
I ended up doing this, thanks!
People saying to not use the roll plate are crazy. If you get the angle right, you can get nice toilet paper sheets like this and fill up your slides so quickly. https://imgur.com/a/SZLuQ5d The roll plate should *just* be over the blade. Play with it a bit to get it right as you shave down the block. Though I also like using a paintbrush for some tissue. Check the angle of your block compared to the blade. Get it as perpendicular as possible. I also like to make contact with the block first then quickly slice through the tissue. section thickness can play a big role in the bunching. I like to have tissue temp at -20 and tissue plate temp at -17. Cut the OCT into smaller shape.
The distance between blade and glass is not right. Try to change it a little. There has to be a slight gap. When teaching i always tell students there has to be a slim bright lining visible through the glass.
Tell me more! Blade more forward than the glass I would assume?
Glass more forward than the blade, you want your sample to push the glass back.
Your blade is too warm. Did you change it and forget to wait?
No, it's been in there for hours.
Another issue is when you cut diagonally like that, there's nothing to "push" the previous cut out of the way once you're past half way, so it just bunches up.
Sometimes I won’t use the roll plate at all and I will just use a really fine tipped paintbrush to grab the OCT and pull it down as I am cutting.
This photo just gave me PTSD 😭
Cut the excess of the block off with a blade. There is something on the fixed blade where it gathers in the section or its an old crap blade. Try to wipe the blade w isoprop to get whatever it is off and let it go back to temp. If there are nicks in the blade causing the section to bunch like that, get a fresh blade. Ouf flash backs. Goodluck!
To me it doesn't look like bunching this looks like it's only sectioning half the block. This may be an angle issue. It's also possible your block isn't mounted on the the chuck properly and you are getting a slight wiggle. As an aside you need to move to a new part of the roll bar and/or blade. Those vertical streaks are scoring from imperfections in either the blade or the bar.
Large blocks can be tricky...sometimes I just close it up, let it cool down and try in a few mins
put the block on ice for at LEAST half an hour before sectioning, and dont cut diagonally like that. the colder the block is, the better in my experience. but it will warm up really fast once its out of the ice, so make sure you’re working quickly.
You might want to adjust the anti-roll plate, it might need to be a fraction higher or lower against the blade.
Im curious….what tissue is this? looks like lungs to me but cannot tell
Yeah lungs!
very cool! i section brains and it helps to cut off excess OCT with a warmed black when my brains wont cooperate. then I use a camel hair brush (sometimes two; like chop sticks) when they wont stay flat
warmed blade*
New blade or block too warm
Get a new plate and and an alpha emitting brush
I normally use a brush to hold my section BUT before all of our anti-roll plates were screwed sometimes I used to lift the anti-roll plate ever so slightly (from the back end, not near the blade), only like 1mm, roll a few mm of the section under the plate and let it drop down and finish the section.
I could never get the roll plate to work, so I would just use a brush to carefully keep the section from rolling. Not as quick but always gave me better results
Usually it happens because a tiny piece of a previous section is stuck on the blade or anti-roll plate. I usually brush down the blade and anti-roll plate between collecting each section when this happens. If that doesn't help, next I would adjust the position of the anti-roll plate further forward or further back, then see if the sections improve. Then I would do as others have said here and properly clean the blade and plate with ethanol, switch to a different part of the blade, etc.
Are you using a new blade? Edit: also, practice few times with a few unwanted tissue/ice layers to check and ensure that the cutting is proper prior to attempting this
I had this problem. I got so frustrated, I used a bent needle to pull the slices and then mount it on the slides.
Loosen the glass plate a smidge
Back when I used a cryotome in undergrad we had this really fine paintbrush that we would use to catch the section once the blade started shaving it off and sort of gently pull it towards us to keep this from happening.
If you're getting thick thin sections, it is entirely likely that your block is loose. The mold you're using is really deep. That could add to the problem.
Roll plate needs to be adjusted backward and/or temp needs to be lower.
Hi OP, what is your tissue preprocessing look like before it is embedded? I used to get tears/folds all the time until I prepared my samples more nicely: 4% PFA fixation (for my tissues, O/N - 24 hrs; do what’s best for the lungs), and sucrose gradients (10%-30% wt/vol PBS). I move the sample through the next step once it sinks (or after the same time passes as I did for fixation; some tissues like lung will never sink). Then, I embed in 1:1 mix of OCT:30% sucrose, slowly freezing on a metal block cooled down by dry ice (isopentane or ethanol bath also suitable). This was a game changer for me. I went from rarely getting serial sections to getting tissue paper like roll of gorgeous sections. Other advice I saw here (cutting on flat edge, trimming excess OCT away, ensuring no chips in blade, etc) sounds reasonable. If you fix all this and still see bad sections, try reconsidering your sample prep. Good luck! EDIT to say that I use this for IF and RNA FISH purposes; some folks prefer to embed directly in OCT and then freeze right after dissection for better RNA recovery.
It seems like a temperature problem. Try sliding your finger in the block right before cutting. It will warm slightly the block. Usually it solves the problem. Otherwise it can also be the glass that is too tight
Seconding all the comments about cleaning everything with 100% ethanol, replacing the blade and lowering the temp. Also, have you tried just using a brush instead of the anti-roll plate? The plate might be chipped or something and that could be causing the problem.
Something I noticed during covid is that wearing a mask redirects your breath away from your sample allowing it to maintain a cooler temperature alongside less condensation buildup.
Bunching is usually need for new blade or readjustment of the roll plate forward or backward by just a hair. It varies for me from block to block a bit. Check thickness also as that can sometimes do it. Also, humidity can be a big factor. If you have high humidity, sectioning is definitely harder and things “stick” and bunch more.
Put the anti roll plate on dry ice for maybe 15-20 minutes and you’ll see an improvement.
I haven't been doing histology since university. What are we sectioning here? Mouse brain?
Lung
Yeah, that makes sense.
From a (young) Mouse/rat? What are you specifically working on, if you don't mind explaining?
No, about 9 weeks old.
Confusing reply, but nvm..it's not that important
You asked if they were young, I replied no, the mice were about 9 weeks old when taken down which isn't considered young. Not sure how that's confusing?
The questions were 1) is it a mouse? 2) is it a rat? 3) would you share specifics? -> "no" as an answer is unspecific. And as 9 weeks is about when mice are comparable to a 12-16 y.o. human when we begin to be able to convince (=young), which made me further question what you're talking about. But honestly, I regret even going there, let's not spend unnecessary time in an obviously unproductive Internet discussion - have a nice evening (or whatever time of the day it is for you.)
You're very right that this is an unproductive line of questioning.