Hi Yun,

I have a suspicion, but it would help if you further described your experimental process from perfusion to slicing.

I'm assuming these are cryosections since 20um is too thin for a Vibratome and rarely seen with paraffin embedding:

How are you cryoprotecting?

How and in what are you embedding?

How are you freezing your brain?

How are you (slightly) warming it before slicing?

And at what temperature are you slicing?

My guess is that these are simply freezing artifacts, but your answers to the questions above will help me or others nail down your issues!

Best,

Alberto

these are most likely freezing artifacts; your cryoprotection is insufficient and may also be generated by insufficient fixation (slower pfa perfusion does not equal to better fixation but rather worse fixation).

Oh, sorry about the minimum words on the experiment process. So we fixed the mice with 4% PFA, which seemed good (they were all nice and stiff) followed by post-fixation over night and then 30% sucrose until it completely sinks to the bottom. We embedded the brains in OCT about 24h before cryosectioning and kept it in -80 overnight. The brains go from sucrose straight to OCT, and only spends about one night in -80.

Hm.. That's weird! If your brains sunk in sucrose they should be cryoprotected. It does sound like you're doing all the right things.

Only other possibility I can think of is the actual freezing step (for example, liquid phase of LN2 will be too fast, while just putting it in a freezer, even - 80, will be too slow).

Isopentane cooled by dry ice, crushed dry ice or the vapor phase of LN2 should all work. I've even seen people use cans of compressed air (turned upside down) to freeze tissue successfully...

Also, I'd recommend that you not waste reagent and time with your IF until you figure this out. That size of artifact should be easily detectable on a completely unstained slice!

Sorry I can't offer further help!

Edit: you could also be slicing too cold, making the blade compress then skip tissue. Such artifacts are usually more elongated holes though, and parallel to the blade.

We embed them in OCT and then freeze the brains in -80. I don’t know, my first instinct when looking at these holes is that there’s a freeze-thaw cycle that’s happening somewhere in the process, even though we don’t realize it. That it’s a temperature thing. But I can’t really say where something went wrong.

If you mean that you just place the embedded brain in the - 80C freezer, I would absolutely try a different freezing method.

I've never tried that method but have been warned that it is nowhere close to the quick, even freezing that's necessary.

Here's my personal favorite protocol briefly if you'd like to attempt it:

After sinking in sucrose, I slightly dry the brain, then drop it into isopentane cooled by dry ice. I never fully embed in OCT, I just use the OCT as a sort of glue to fix the tissue to the chuck.

Alberto Sepulveda Rodriguez

Yeah, I hear that liquid cooling is better for the brains. Thanks for the tip.

Dear colleague:

In our laboratory we experienced similar problems, when after the criosectioning, we stored the brain slices in an inadecuate criopreservative solution. If this solution forms crystals, you are going to find holes in your slices for sure.

Our protocol is slightly different: We bring out the brains from -80°C, to -20°C, at least for an hour, the we embebed the brains in OCT and perform the cutting. The obtained slices are stored at -20°C in cryopreservative solution. For detailed composition of cryopreservative solution, please see: Article Chronic Stress Triggers Expression of Immediate Early Genes ...

Good luck!!!!!

Ah, another detail I forgot to mention. These brain slices are attached to the slides after cryosectioning, not preserved as free floating sections in cryoprotectants.

Dear colleague:

If you stored your attached slices at -80°C, you must take them out only when you are going to procces them. Just that day you must change directly the coverslides to -20°C for at least 20 min, then change then again to 4°C for at least 20 more minutes. After all that your samples will be safely unfrozen and ready to work in the tablelab.

Good Luck!!!

I have had problems with holes in cryosections and, at least in my case, the problem was indeed insufficient cryoprotection.

One way of troubleshooting is to try shaking your container with brain + sucrose solution after the brains sink. Sometimes the brain floats again, indicating that it is not completely equilibrated with the sucrose. I would also recommend doing this step overnight, if you are not already doing so.

Lastly, make sure you have a sufficiently large volume of sucrose. I would say 4ml/brain is a safe volume.

Best of luck!

The attached slides were left out to dry in room temperature for a couple hours after sectioning and then stored in -20. Thanks for the tip Ronan. I feel that it’s a temperature thing, as only temperature stuff would cause this “frozen tofu” effect.

Hi Yun,

First thing i would check is where the holes are. If they are primarily in the center of the tissue then its improper cryoprotection. Remember that sinking in sucrose is dependent on density of the brain being same as the sucrose solution and is only an indicator of cryprotection. One solution is to take it through a gradient of sucrose. 10% 1h - 20% 1h - 30% until it sinks. This gradual increase of concentration allows for better perfusion of the cryoprotectant.

The other issue, as someone mentioned before, is it being too cold when you cut. Usually embedding in OCT is best done in dry ice and not by putting it in -80. Brains stored at -80 post embedding should be allowed to stand in the cryotome for an hour or so at -20 (I am assuming you are cutting at -18 to 20 degrees) before cutting to equilibrate the temperature of the brain block to the cutting temperature. Otherwise you brain is too cold and will crack on sectioning, as the temp difference between the blade (-20) and the brain (~ -80) is too large. Finally make sure you are using the right blades. For thin sections (10 to 20 um) I get better results with disposable blades as they are super sharp and cut through the brain like a hot knife through butter. Reusable knives, even if sharpened, dont have the sharpness of the disposable blades to give you thin tissue and ofthen instead cause damage.

I cant give more advice as I am unable to open the image file you sent. Its always good to send a tiff or jpeg file for these purposes as most image viewing softwares support these file formats.

Hope it helps!

Hello, since this question is referenced on google I would like to share my experience even if it is late to reply to the original poster, hoping this can help someone looking for this answer in the future.

I had some similar holes in cryosectioned brain slices. This happened for brains perfused in PBS only and also for brains perfused with PBS + PFA 4%. In my experience, holes are mainly caused by poor fixation (during and after perfusion) and slow freezing/thawing.

Eventually, after several shots, I came up with a protocol that improved the quality of the slices and avoided holes.

I made a custom embedding chamber by cutting a 15 ml tube, then placed in a 50 ml tube cap. See attachment.

1. prefuse the mouse (PBS or PBS + PFA 4% depending on necessities)

2. fix the brain overnight in PFA 4%

3. wash the brain with PBS

4. cryopreserve brain in filtered sucrose solutions: start with 10%, when it sinks remove sucrose 10% and add sucrose 20%, when it sinks remove sucrose 20% and add sucrose 30%.

5. when it sinks remove sucrose 30% and place the brain on paper to absorb excess sucrose

6. if possible, use a scalpel blade to create a flat surface on the back of the brain by removing the cerebellum

7. put dry ice in a plastic box and place a 100 ml becker in the center

8. pour ~50 ml of isopentane at -80 °C (the final temperature should be between -40 and -20 °C) in the becker

9. pour ~5 mm of OCT at the bottom of the chamber and place the brain (olf. bulbs facing up) in the OCT

10. pour OCT to cover the brain

11. by using forceps, take the chamber from the cap and submerge it in the isopentane

12. after 2-3 minutes, remove the chamber from isopentane and store at -20 °

13. for cryosectioning, move the whole chamber from the freezer to the cryostat chamber (previously set at -20 °C) and disassemble the chamber.

This protocol allows snap freezing of the brain without a strong temperature shock. Also, the brain is stored in OCT directly, thus avoiding the thawing caused by pouring OCT at room temperature when the brain is frozen.