Tissue is full of holes

[PennPostDoc]

I am perfusing P45-P90 C57B6/J-129 hybrids with PBS followed by 4%PFA pH 7.3.  I dissect out the brain and olfactory bulb, post fix in the same 4% PFA for 24 hours, and cryoprotect with a 10-20-30% sucrose PBS gradient over 2 days. 

For my PFA solution:

4 g PFA dissolved into 85 ml dH2O and 5 drops 10N NaOH, heat and stir.

When clear, cool to 4C, and add 1M Phosphate buffer, 1ml 0.5M EGTA, and 200 ul 2M MgSO4.  I pH to exactly 7.3 and I then filter the PFA.  I use pentobarb to anesthetize the mice, perfuse with PBS, and then use 12-15 mls to SLOWLY perfuse and fix.  Dissect, post fix ect. 

After cryoprotection, I freeze the brains at -80C in OCT embedding, and section at -20C on a cryostat.  anywhere from 10-20 um sections. 

After I air dry the sections for 1 hour, and dip in 4% PFA to post fix, I then wash in PBS for 5 min.  When I examine the tissue under a microscope, there are holes EVERYWHERE.  Cortex, olfactory bulb, amigdyla ect.  Even after fully processing for IHC or classical stains, the neurons look like they are ripped away from the EC matrix, and there are just holes all over the place. 

I had the same problem at the tail end of my Ph.D., and it is continuing now in my post-doc.  I just sectioned 10 animals, and they are ALL ruined. 

Can anyone give me an idea of where the holes are coming from an how to fix this issue.  I just bought a Luxol blue/Cresyl Violate myelin-Nissel stain kit that is expensive, and my test slides for staining look absolutely awful.  I am not a newbie at sectioning by any means, but no one at Penn can seem to give me an idea of where the problem is. Any help is most appreciated. 

[richard03]

I am not sure if the PB buffer cause this, but "1M Phosphate buffer" seems too concentrated. It should be 0.1M.

Saline (0.9% NaCl) is normally used for rinse blood before fixative when doing perfusion. I never used PBS.

After remove brain, are you sure the brain is fixed well?

Most important step is freezing brain. You should not freeze brain in -80 and then embedding in OCT. If you did that, this was probably main reason for the holes.

You should either quickly freeze brain with liquid nitrogen or other freezing agent or place OCT to cover brain BEFORE put in -80 freezer.

Hope this helps.

[PennPostDoc]

I am not sure if the PB buffer cause this, but "1M Phosphate buffer" seems too concentrated. It should be 0.1M.

I should be more specific. 

4 g PFA dissolved into 85 ml dH2O and 5 drops 10N NaOH, heat and stir.

When clear, cool to 4C, and add 10 mls of 1M Phosphate buffer, 1ml 0.5M EGTA, and 200 ul 2M MgSO4.  I pH to exactly 7.3  adjust volume to 100 total mls and I then filter the PFA

So in the end, there is 10 mls of 1M phosphate buffer in 100mls TOTAL volume, which makes it 0.1M at final dilution.

I just looked at more sections I did the other day, and they are truly awful.  There are holes upwards of 50-75 micros in the tissue.

Additionally, the sections refused to come off the knife flat, and there are a great many folds and kinks in the tissue, which is telling me that the blade is "jumping" as it cuts through the tissue.  This is a big sign to me that there are already holes in the tissue, and not caused by sectioning and melting to the slide. 


What I have found is that by embedding the brain in a cylinder (a soft plastic tube  1.4 cm in diameter cut to 2 cm long) that my sections come off nearly perfectly.  The blade enters the leading edge of the cylinder and cuts smoothly all the way through.  There usually is not folding of curling.  HOWEVER, it has proven a real nightmare to be able to freeze the brain quickly, while maintaining the tissue orientation the way i need it to sections coronally. 

Normally, I put the cylinder on a metal plate on dry ice, and put a small bit of OCT at the bottom as a base.  I then put the brain standing vertically in the center of the tube so the cerebellum (which I dont care about) sticks to the OCT and holds the brain totally vertical, and fill the tube with OCT completely.  I then put the tube in the -80C freezer until it is frozen solid. 

Here is a crude drawing of what I am trying to explain. 

[excalibur]

24 hours is not sufficient to fix a whole brain. Increase to at least 48 hours.

Your sucrose protocol sounds good. Did the brain sink before freezing?

You are freezing a large mass too slowly and this is causing the holes.

Try finding a smaller container like a Peel-A-Way mold.

Also, if you are fixing the tissue, why not just do paraffin? This would eliminate all of your current problems.

[CanuckPhD]

24 hours is more then sufficient to fix a perfused mouse brain. In fact I often only fix for 2 to 4 hours and have great morphology and good IHC.

The holes are coming from the fact that you are freezing your tissue in the -80. This should never be done as it leads to exactly the problems you are finding in the tissue.

This is our method to freeze brains and results in good morphology. I have been doing this for several years and am almost always happy with the results.

1. perfuse rodent with 0.1 M PBS and 4% PFA (5 minutes each, 10 ml/min rat, 7mls/min mouse)
2. place tissue in 4% PFA for 2 to 48 hours (depending if free floating required)
3. after fixation place in 15% sucrose for 24h
4. replace sucrose with 20% until tissue sinks (24h usually) NOTE: I find that 30% makes the tissue more sticky and that 20% gives good results. In fact for brains I usually just replace with another 15% sucrose.
5. remove tissue from sucrose, blot off any excess and place in mold. This is an important step to avoid a "shell" of sucrose which coats the tissue and slows the freezing process.
6. cover with OCT
7. place mold in -60 cooled isopentane (put isopentane in plastic container and place in a bath of dry ice and absolute ethanol)
8. freeze for 1 minute
9. remove and place on dry ice for an additional 15-30 minutes
10 store in -80 until required

NOTE: it is important that the isopentane is -55 to -60. Any colder and tissue become brittle, and warmer and you get holes.

Good luck

[Cardio]

Canuck is right. You are fixing fine but you are freezing to slow. If you can I would  suggest post-fixing if your experiments allow it. This would be on top of cyroprotecting and freezing faster.

[nzkat]

Hi - we had a similar problem in our lab - for us it was due to the failure of the cryoprotection step. Make sure your brains sink properly in the 30% sucrose. This may require you to change the sucrose a couple of times. We then freeze the brains quickly with dry ice and cut on a microtome for free floating immuno
Kat

[pandanac]

In continuation with the previous posts regarding the holes... my situation is the same however i am using peelaway mold, but freezing in -80. The tissue is well perfused in pbs and 4% PFA and post fixed in PFA for  24 hrs and then transferred to 30% sucrose, till it gets precipitated.
The Peel-A-way molds which we had purchased is good and very user friendly.Is the peel away mold taking more time in the -80 
I usually use to freeze in -80 freezer, using a chewing gum wrap (The one we get with Orbit..etc; sounds crude but it works well.). With this new Mold that I am using sometimes the tissue is good, but mostly it is not. I am having horrible time and I need your help in this regard. Should I use liquid nitrogen to freeze the brain in OCT using the mold, as the -80 is probably taking more time to freeze the OCT with the brain...


Please help.. me kindly...

[disco volante]

For what's its worth, we follow a fairly similar protocol, but we never embed our brains or freeze them at -80.  We keep them at 4C in 0.1M PB with 0.1% sodium azide until we're ready to use them.  When we have finished cryoprotecting, we stick a block of brain to the cassette with a drop of OCT, then freeze with histofreeze.  Since the brain is small and not embedded in anything, it freezes pretty much instantly.

[pandanac]

hi,
Thanks for the reply. Well the brain I am using is adult rat brain, 1 year old. and i need to freeze atleast 80% of the brain..(I only remove the forntal cortex and a little bit of cerebellum[I dont need the pons-medulla])

So the brain is not small, it pretty big and is taking atleast 10 min (max) to freeze

[elfrimml]

Freezing too slowly can cause holes in the tissue.  I would suggest freezing in your tubes, but with liquid nitrogen.  If you do this it won't take 10 minutes for anything to freeze and you'll get a whole lot less freezing artifact which causes holes in the tissue.