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Author Topic: Help with freezing artifacts from fixed adult rat brain!  (Read 3562 times)

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Offline LAngie

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Help with freezing artifacts from fixed adult rat brain!
« on: February 10, 2015, 01:16:31 PM »
Hi, All
First, thanks for reading my thread. I am new to both IHCworld and histology.

My crystal violet staining showed all my rat brain sections (14micron, throughout a brain) have many holes or freezing artifacts.

Here is how I treated the brain. I perfused the rat with saline and 4%PFA, extracted the brain and left in 4%PFA overnight. Afterwards, the brain was transferred into 10%, 20% and 30% sucrose. I confirmed the brain sank in each solution. I left the brain in 30% sucrose for about a month before I freeze it. When I freeze down the brain, I cutoff the cerebellum and embedded the brain in the tissue-tek mold with OCT. Then I emerge the entire mold into dry-ice/acetone chilled isopentane. Sometime I saw bubbles but a lot of time i didn't. I waited for a minute or so before I took out the brain, I wrapped it in foil and left on dry ice. After I freeze down all the brains, I transferred all samples into -80C. One night before sectioning, I transferred the brain into -20. I sectioned at -18 Celsius degree.

I should clarify that I used 20 weeks old SD rat brain and my section is 14 micron thick, this is what I have to do for the project.

The artifacts I saw are all over the sections, and it looks like mad cow disease if that is an appropriate description.

My problem is that I freezd down all my 80 rat brains and they might all have crazy artifacts. So no turning back.

I heard from other people that I can thaw and refreeze my brains and I wonder does anyone know how to do this. My understanding is that I thaw my brain in either 4% PFA or 30% sucrose and refreeze again. I heard that it will allow the crystal formed during the first freezing melt and give the brain a second chance to be properly freeze again. I also wondered what is wrong with my previous freezing process. Someone suggested that my isopentane is not cold enough if it is only chilled with dry ice/acetone.

In summary, my two questions:
1) What is wrong with my freezing process?
2) Would I be able to refreeze my brains to save them from crazy artifacts? How to refreeze in my situation?

Thanks very much in advance!

Angie

Help with freezing artifacts from fixed adult rat brain!
« on: February 10, 2015, 01:16:31 PM »

Offline gula

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Re: Help with freezing artifacts from fixed adult rat brain!
« Reply #1 on: February 10, 2015, 01:43:57 PM »
Sorry, I have no really good answer for you. Is it an usual process in your lab to let the tissue for such a long time in 30% sucrose before freezing? Did you store them in the fridge during this month? For my ears it sounds a long time and I bother, if brain-tissue is preserved well in this way.

How do the holes look like? Cutting artefacts like chatter or clear round holes like vacuoles? For cutting-artefact I would try to cut at higher degrees, or warm the surface shortly with the warm finger (with gloves :)).

In my experience bad frozen tissue never gets better after thawing and refreezing. The crystals have destroyed the tissue-matrix and that will never get repaired again.
If you have the possibility to continue your experiment with paraffin-sections, you can thaw the pieces in 4% NBF, let them fix for an additional night and go on with paraffin-processing.

Offline LAngie

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Re: Help with freezing artifacts from fixed adult rat brain!
« Reply #2 on: February 10, 2015, 05:20:35 PM »
Hi, Thanks very much for your reply.

I always kept my PFA and sucrose solutions with brains inside in plus 4 fridge. It is good know that it doesn't worthy to thaw and refreeze. I thought I had a chance to rescue my samples but I guess not.

I do think those are frozen artifacts as they are round holes and I had experienced people looking at them.

Thanks,
Angie

Re: Help with freezing artifacts from fixed adult rat brain!
« Reply #2 on: February 10, 2015, 05:20:35 PM »