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Author Topic: Preparation and cutting of OCT embedded mouse brains  (Read 21281 times)

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Offline nords21

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Preparation and cutting of OCT embedded mouse brains
« on: April 08, 2009, 07:33:55 PM »
Hello everyone,

For the last few months I started working in an EAE model which requires a great deal of sectioning/staining in brain, spine and some other organs on occasion.  Most tissues come out fine when I cut then except for the brain.  I typically see the fallowing things; the tissue folds and air bubbles get caught under the sections all over, also the brain tears and has a lot of holes in it sometimes. I notice when the tissue passes over the blade, the sections seem to be under more pressure or stress than I normally see with other tissues and its "crunches" up and rolls really bad.  The tissue also separates from the OCT a lot and the holes and tares are noticeable while cutting.  I have tried different blades, I mostly use Accu-Edge though; different temperatures ranging from -15 to -25 for both chamber and object temperature and different section thickness, ranging from 4 micros to 25 microns.  No matter what I do they always look this way. In turn the staining looks horrendous.

I acquire tissues after perfusion with PBS then 4% PFA, then store for 24 hrs at 4 C in 10% formalin.  After 24 hrs I place them in 30% sucrose made in 1x PBS (I have tried higher concentrations 45% and 70%, with no noticeable difference).  Then I embed the tissue in OCT using dry ice and 2 methyl butane.

If anyone has any experience with frozen mouse brain and could help I would greatly appreciate it.

Best,
Norbert

Preparation and cutting of OCT embedded mouse brains
« on: April 08, 2009, 07:33:55 PM »

Offline nords21

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Re: Preparation and cutting of OCT embedded mouse brains
« Reply #1 on: April 09, 2009, 05:37:15 PM »
Bump to the top for help.

Offline Cardio

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Re: Preparation and cutting of OCT embedded mouse brains
« Reply #2 on: April 11, 2009, 02:31:16 PM »
First off I don't have much experience with brain tissue but from my experience with other tissue I can say that bubbles form more with pre-fixed tissue. Not sure why but even if you are very good at taking up the sections you can still get bubbles and wrinkles form. I speculate that the bubbles/wrinkles form because the tissue is often stiffer and less pliable when fixed/frozen.

Here are a couple of things you could try.

1. Fix in 4%PFA overnight and then cryoprotect/freeze. Post-fix slides.

2. Blot your tissue more on a kimwipe. Alot of times any residual pbs/sucrose will cause the blade to drag on the tissue when sectioning.

3. I doubt this is the problem but try isopentane (2-methlybutane) cooled with liquid nitrogen.

« Last Edit: April 11, 2009, 02:37:24 PM by Cardio »

Offline nords21

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Re: Preparation and cutting of OCT embedded mouse brains
« Reply #3 on: April 13, 2009, 02:27:53 PM »
Thanks for your suggestions. I will try to dry my tissues on a kimwipe a little more before embedding.  I may try to cut some non-fixed tissue to see if I find a difference with bubbles and wrinkles.


Offline cincanada

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Re: Preparation and cutting of OCT embedded mouse brains
« Reply #4 on: April 14, 2009, 12:52:48 PM »
What are you cutting your tissue on?  A cryostat?  I can slice rat brain up to 10 microns and mount onto a slide quite easily.  I slice at -25 but before each slice I run my thumb over the mold, when you slice it I find it does not curl as much or at all as when I do not.  The bubbles could be from the OCT when you build your mold.  What do you do when you do this step?  How big are your tissues before you cut?  Lastly, do you cryoprotect you tissue in 30% sucrose?  Before you process do you make sure the tissue has sunk to the bottom.  Do not put it in anything else after the sucrose other than OCT.  The sucrose removes any water left in the tissue which will help prevent holes in the tissue.


Offline CanuckPhD

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Re: Preparation and cutting of OCT embedded mouse brains
« Reply #5 on: April 15, 2009, 02:25:47 AM »
I have been using 20% sucrose instead of 30% and found the cutting easier and the morphology is fine. We were having problems getting sections with the 30% due to curling of the sections.

As mentioned above it is critical to remove all the sucrose from the surface of the brain. I use a kimwipe and gently blot it away.

Offline nords21

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Re: Preparation and cutting of OCT embedded mouse brains
« Reply #6 on: April 17, 2009, 01:42:19 PM »
Thanks a lot for all your replies.  I am using a cryostat (leica 3355S) I have cut from ranges of -15 to -25 at 4 micron to 20 micron with everything from 30% sucrose fix to 75%.  I am going to give the 20% sucrose a try (thanks Canuck) - as well as a suggestion from a colleague to try a 30-70 mix of 20% sucrose and OCT to actually embed in.  The tissues all do sink, yes. The only ones that have issues are the 75% sucrose ones. They never really sink all the way (let it run for 1 month in 4degree)

And the bubbles are not from the OCT, rather from sections of tissue that dont touch down to the slide.  There isnt enough tissue for it to fold on itself, but the tissue is free enough to form these bubbles. When I treat the tissue for staining the bubbles often tear away and leave a hole in the tissue.

Offline Cardio

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Re: Preparation and cutting of OCT embedded mouse brains
« Reply #7 on: April 17, 2009, 02:34:25 PM »
Nords21- Whats the rational behind 75% sucrose/1xPBS. Normally most people displace the water in the tissue with 30% to 20% sucrose/PBS solution and have very good morphology. The 75% probably won't sink because of the buoyancy. You can't sink something that is lighter/less dense than the surrounding solution.

I have sectioned using a 50/50 mixture of sucrose/1xPBS and OCT. IT worked ok but the sections react to the temperature in cryostat differently from 100% OCT.

The bubbles are a result of how you are taking up the sections. IF your slide is parallel to the section and the section is not flat you will have bubbles form. IF you put a slight tilt to the slide when you approach the section you will limit the bubbles. In essence you are stretching the section but very slightly. This is not as easy as it sounds but I would ask someone with a lot experience to show you 1st hand on how to pick up sections to limit bubbles.

THe other way to limit bubbles is to have a very flat section. This involves the setup of the cryostat. Correct placement of the anti-roll plate (Glass plate that keeps the sections from rolling) The angle of the knife and the temp of the cryostat.

« Last Edit: April 17, 2009, 02:36:30 PM by Cardio »

Offline hgurji

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Re: Preparation and cutting of OCT embedded mouse brains
« Reply #8 on: April 29, 2009, 04:53:03 PM »
Hello All,
I have a question regarding the length of time a sample can be kept in OCT at -80C and still be a viable sample for doing IHC?  I currently have various organs (skeletal muscle, kidney, lung, liver) that I place in OCT and freeze in liquid nitrogen and keep in -80C.  Is there a length of time that these samples can be kept frozen before tissue integrity or antigenicity starts to degrade while in the OCT?  Thanks for your help in advance.

-hgurji

« Last Edit: July 09, 2009, 11:01:42 AM by Cardio »

Offline alicem

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Re: Preparation and cutting of OCT embedded mouse brains
« Reply #9 on: May 11, 2009, 10:42:12 PM »
Dear Norbert,

I have experienced the same problems but recently discovered that if you place your tissue overnight in 30% sucrose and PBS, then the next night in 50% sucrose PBS and 50% OCT, then the next night in 100% OCT and I even leave my tissue maybe 2-3 nights in OCT (in the fridge)... this gives plenty of time for the OCT to infiltrate into all the cracks etc.
When you freeze it make sure there are no air bubbles in the OCT particularly around the tissue as this will cause folds etc. Also make sure there is no water or PBS present when freezing but there shouldn't be if you have left your tissue in OCT for a while.
Make sure the freezing temp you are cutting at is sufficient.  I cut unfixed frozen sections at 12um and have no problem... so fixed tissue should be better.
Hope this helps

Offline Cardio

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Re: Preparation and cutting of OCT embedded mouse brains
« Reply #10 on: July 09, 2009, 11:01:59 AM »
Hello All,
I have a question regarding the length of time a sample can be kept in OCT at -80C and still be a viable sample for doing IHC?  I currently have various organs (skeletal muscle, kidney, lung, liver) that I place in OCT and freeze in liquid nitrogen and keep in -80C.  Is there a length of time that these samples can be kept frozen before tissue integrity or antigenicity starts to degrade while in the OCT?  Thanks for your help in advance.

-hgurji



I haven't seen any conclusive study analyzing what your question is driving at so you will probably get varying opinions.
Here are some things to consider.
1. IS the tissue fixed before freezing and with what fixative/length of time. Total fixation can also mean a loss of antigenicity hence antigen retrieval.
2. Type of tissue. Not all tissue will perserve the same.
3. Protein of intrest. Are you looking for a easily degraded protein or is it a hardy structural protein.
4. Here is probably the most important. How are you storing the OCT/tissue.
- IS it vacuum sealed to prevent drying out (freezer burn), is the tissue stored in screw cap falcon tube or is it stored in a plastic sandwich bag.

All of this will determine proper storage length. In my experience (mainly heart tissue, I have had no loss of antigenicity in 1 year old tissue that was not fix prior to freezing. With tissue fixed before freezing I have good results with tissue stored for roughly 3 years.

Hope that helps.
« Last Edit: July 09, 2009, 11:03:40 AM by Cardio »

Re: Preparation and cutting of OCT embedded mouse brains
« Reply #10 on: July 09, 2009, 11:01:59 AM »