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Author Topic: Snap Freezing PFA fixed and Sucrose cryopreserved brain  (Read 17891 times)

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Kaushik.shah

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Snap Freezing PFA fixed and Sucrose cryopreserved brain
« on: March 16, 2009, 05:03:53 PM »
Hi all:

Can anyone suggest whether to snap freeze brain directly without embedding in OCT will give good sections?

Protocol: Intracardially PBS perfusion, extract brain out and cut into 1 mm sections on tissue chopper. As I want to have this tissue stained for TTC first which needs live tissue. Then I fixed the slices into 4% PFA and 20%-30% sucrose cryopreserved. I embed them into OCT carefully in order to have slices flat, but sometimes during fixation slices acquired some curves, thus always is not a good luck to get the flat slices embedded in OCT after freezing, even after due care.

So, I was kind of thinking of Freezing the tissue without embedding in OCT following fixing and cryopreserving. I had previously tried freezing directly fresh tissue from TTC using Isopentane, but doesn't turnout good when cutting 20 micron section.

Highly appreciated if someone gives some tips on problem or some new method to accomplish this.

Snap Freezing PFA fixed and Sucrose cryopreserved brain
« on: March 16, 2009, 05:03:53 PM »

Offline Cardio

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #1 on: March 16, 2009, 06:00:02 PM »
Not sure exactly what your question is? Do you mean not using OCT at all and trying to section the sample? I have never tried that but it seems like it would be very hard.

I don't work with brain tissue but if the problem is the curvature of the tissue have you tried anything to correct this? For example if you are using a round bottom tube to fix in have you used a 6 well plate which would be flat or something to apply pressure to keep the tissue flat. I have done this with hands and it works well.

Next have you tried embedding the tissue in oct then freezing with isopentane?

« Last Edit: March 16, 2009, 06:02:08 PM by Cardio »

Kaushik.shah

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #2 on: March 16, 2009, 11:10:15 PM »
Not sure exactly what your question is? Do you mean not using OCT at all and trying to section the sample? I have never tried that but it seems like it would be very hard.

I don't work with brain tissue but if the problem is the curvature of the tissue have you tried anything to correct this? For example if you are using a round bottom tube to fix in have you used a 6 well plate which would be flat or something to apply pressure to keep the tissue flat. I have done this with hands and it works well.

Next have you tried embedding the tissue in oct then freezing with isopentane?


Thanks for the reply, what I meant is I am slicing the brain to stain it with TTC and I want to use same TTC stained brain slice for further immunohisto or immunofluroscence. I have been Slicing the brain in 1mm for TTC stain and then fixed them in PFA and cryopreserved in sucrose and then embed in OCT with due care to have the 1 mm slices lying flat in mold while freezing. I do get good sections with this, but the problem is I get almost 25-30 sections per slice and if slice is not flat, which happens sometimes in my case after fixing PFA and that really knocks out my 10-15 section untill I adjust it to get the tissue as flat as possible while cryosectioning.

So, my question here is can we proceed freezing fixed and cryopreserved brain slices directly in isopentane without embeding in OCT. Does this will give good sections?    Or can anyone suggest me how we can freeze the fresh tissue after TTC stained Brain slices.
I just want some alternative in order not to loose my sections in getting tissue flat as possible.

Thanks for help

« Last Edit: March 16, 2009, 11:13:18 PM by Histocraz »

Offline Cardio

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #3 on: March 17, 2009, 09:25:54 AM »
I have done what you are suggesting albeit heart tissue (liquid nitrogen) and I noticed a slight thaw of the tissue. It also rolled away from the OTC a bit more while sectioning.

If the goal is maximizing the amount of slides you can produce I can offer one suggestion.

As I said before I work with heart tissue which is sometimes vibratomed into 300 micron sections then embedded for 10 micron sectioning.

The procedure for the mold: (You need 4 non-charged slides + cryofreeze can)
The section (pre-soaked in OTC) is placed in the middle of the slide with room to spare around the sides. The next 2 slides are act as spacers to keep the thickness. They are placed perpendicular (Top and bottom) of the sections but not against the sections.

Next add one small drop of OTC above and below the section.
Then take the last slide and place it on top of the sections (parallel to bottom slide).

With your figures still pressed agianst the slide take the cryofreeze can and freeze sample. (The spray can freezes at -51c). IT takes 5-10 seconds to freeze sample.

The sections are now placed on a pedestal that has a large amount of OTC frozen to it. One side note, the frozen OTC is shaved and in line with blade.

Next the sections are casted on the OTC and pedestal.

It is really not that hard and if you try it I can offer some more suggestions + pics.



« Last Edit: March 17, 2009, 12:13:09 PM by Cardio »

Kaushik.shah

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #4 on: March 17, 2009, 04:56:46 PM »
I have done what you are suggesting albeit heart tissue (liquid nitrogen) and I noticed a slight thaw of the tissue. It also rolled away from the OTC a bit more while sectioning.

If the goal is maximizing the amount of slides you can produce I can offer one suggestion.

As I said before I work with heart tissue which is sometimes vibratomed into 300 micron sections then embedded for 10 micron sectioning.

The procedure for the mold: (You need 4 non-charged slides + cryofreeze can)
The section (pre-soaked in OTC) is placed in the middle of the slide with room to spare around the sides. The next 2 slides are act as spacers to keep the thickness. They are placed perpendicular (Top and bottom) of the sections but not against the sections.

Next add one small drop of OTC above and below the section.
Then take the last slide and place it on top of the sections (parallel to bottom slide).

With your figures still pressed agianst the slide take the cryofreeze can and freeze sample. (The spray can freezes at -51c). IT takes 5-10 seconds to freeze sample.

The sections are now placed on a pedestal that has a large amount of OTC frozen to it. One side note, the frozen OTC is shaved and in line with blade.

Next the sections are casted on the OTC and pedestal.

It is really not that hard and if you try it I can offer some more suggestions + pics.


Thanks for suggestion. Could you please send me some pics so I can better understand and try the way you are suggesting.
I appreciate if you have any other suggestions on for freezing the brain slices (fresh or fixed)

Would you also please tell me which spray can you use. I saw TBS freezing can which are kind of bit costly, and the one from Decon, which comparatively cheaper. Which one do you recommend.


« Last Edit: March 17, 2009, 05:57:51 PM by Histocraz »

Offline Cardio

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #5 on: March 18, 2009, 01:21:12 PM »
I use cytofreeze from VWR cat# 16650-029. $70.00 for a case of 6 cans. One can should freeze well over 10 slices.

I will get back to you on the pics and further description.

Do you get flat 1 mm sections? This could also be a problem. I  know on a vibratome its easy to go to fast and curve the tissue which fixation only enhances.

This is a video of another method to get samples flat.

http://www.pathologyinnovations.com/Face%20down%20cryoembedding.htm

My concern with this method is that the temperature for fixed tissue needs to be as cold as possible (cryostat is bad). Secondly, If you look at the aluminum block he froze at the very in of the movie it has remnants of tissue. This also can happen with the slide method I told you about but you can use a thin film of silicone grease to get rid of this problem. Basically you apply a small amount of grease and wipe it completely off with a kimwipe. The grease is not autofluorescence.

You should also look at the paper method which may prevent this problem.

http://www.pathologyinnovations.com/Paper%20Embedding.htm
« Last Edit: March 18, 2009, 04:16:13 PM by Cardio »

Offline Cardio

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #6 on: March 24, 2009, 11:55:10 AM »
Here are the pics.

The spacers are coverslips with the tape. You can change the thickness to what ever you like by adding more coverslips or use slides.

« Last Edit: March 24, 2009, 01:56:32 PM by Cardio »

Offline Cardio

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #7 on: March 24, 2009, 12:04:03 PM »
Sandwich the tissue with another slide and while pushing down with your fingers freeze until OTC is white (10-15 secs). Be sure to remove your fingers after 3-5 secs. You can also double up on the gloves so you don't hurt yourself. Next place the sample in -80c for .5 to 1 hour.

After 1 hour take the sample out and pinch the 2 slides to remove the top slide. Next remove the spacers. Normally you can just rotate them in toward the OTC.

« Last Edit: March 24, 2009, 01:57:03 PM by Cardio »

Offline Cardio

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #8 on: March 24, 2009, 12:10:29 PM »
Cut a 50 ml falcon tube and place the flat portion in the cryostat.

Fill it with OTC and let it freeze. Next take it out of the tube and freeze it on the pedestal.

Shave the OTC down until it is even with the kinfe.
« Last Edit: March 24, 2009, 01:33:17 PM by Cardio »

Offline Cardio

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #9 on: March 24, 2009, 01:40:55 PM »
Now you will freeze the sample to the large block of OTC.

The setup is pretty generic but the point of the 2 vertical slides is to keep the sample in plane with the knife.

To freeze the sample to the block place a fair amount of OTC on the block unit it starts to run down the block. Next take your frozen tissue on the slide and place it on the block.

Very quickly freeze the sample to the block with the cryofreeze can. Main point here is to not thaw your tissue with warm OTC.

After 10 minutes remove the slide by gently pulling on one side. IF it doesn't come off you can warm the slide with your thumb to loosen it.

I didn't have time to edit the pics but if you have any questions let me know. Best of luck.

« Last Edit: March 30, 2009, 11:23:02 AM by Cardio »

Offline ImmunoNYC

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #10 on: March 25, 2009, 09:52:12 AM »
Cardio, what is the  advantage of your technique over just freezing heart or sections of heart (or brain) in a mold and sectioning it? I am confused and it sounds like a lot of work so I want to know what is the main purpose? Thanks.

To the OP, if you want to get every brain slice why not fix, cryoprotect then freeze in a large mold (like a peel-a-way mold)? I use a metal tray floating on liquid nitrogen in a styrofoam box with a lid.

Take the brain (if fixed, cryoprotect in 30% sucrose/PBS, then "wash" sucrose away in a petri dish filled with OCT or if fresh just use directly).  Place a small amount of OCT in bottom of mold and place on freezing tray. Allow it to start freezing but not freeze entirely (layers of completely frozen OCT can separate and cause thick-thin sections etc). Then remove half frozen OCT from freezing chamber and quickly place whole brain into mold. Fill with OCT. Place back into freezing chamber and replace lid. Voila, in a minute or two the block is frozen and can be sectioned in any orientation. Wrap in foil and store at -80 deg C.

Offline Cardio

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #11 on: March 25, 2009, 02:10:15 PM »
So I primarily use this technique to screen a large amount of grafted hearts. The tissue is first fixed then vibratomed into 300 micron sections then screen for grafted cells via marker egfp. The positive sections are then frozen in this manner. 

The advantage is that you maximize the amount of slides with correct orientation. You are right that you can embed the tissue in another manner and still get good sections. However, Kaushik wants to have perfectly flat tissue
so he doesn't under represent a area of tissue, alteast this is my interpretation of what he wants. The technique is also good for flattening tissue that has been curved slightly or for digits of a hand/foot.
« Last Edit: March 25, 2009, 02:59:39 PM by Cardio »

Offline ImmunoNYC

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #12 on: March 25, 2009, 08:54:52 PM »
Interesting, thanks!

Offline immunofluor

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Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #13 on: March 26, 2009, 11:26:54 PM »
So I primarily use this technique to screen a large amount of grafted hearts. The tissue is first fixed then vibratomed into 300 micron sections then screen for grafted cells via marker egfp. The positive sections are then frozen in this manner. 

The advantage is that you maximize the amount of slides with correct orientation. You are right that you can embed the tissue in another manner and still get good sections. However, Kaushik wants to have perfectly flat tissue
so he doesn't under represent a area of tissue, alteast this is my interpretation of what he wants. The technique is also good for flattening tissue that has been curved slightly or for digits of a hand/foot.

Thanks Cardio, I will be trying your technique next week and will keep posted if any problem arises.

yeah you are right, for 1mm thick Brain slices after fixing them in 4 % PFA and cryopreserving in 20 and 30 % sucrose.....I want the slices to be really flat for the cryosectioning.

I guess...your techniques should work for me...

thanks again for your kind information.

Re: Snap Freezing PFA fixed and Sucrose cryopreserved brain
« Reply #13 on: March 26, 2009, 11:26:54 PM »