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Author Topic: Incomplete Penatrance with Paraffin Embedding  (Read 4942 times)

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Offline YouDirtyRat

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Incomplete Penatrance with Paraffin Embedding
« on: April 18, 2010, 05:22:28 PM »
I am encountering a very frustrating problem recently with 6 week old mouse brains not completely embedding with paraffin. A summary of my protocol is as follows:

1. Perfuse mouse with 60ml PBS. Dissect brain
2. Place brain into Neutral Buffered Formalin at room temperature overnight.
3. The next day, make midsaggital slice of brain and place in 70% IPA. Proceed with tissue processing.
4. My tissue processing protocol is pretty standard, i.e. the normal dehydration process and then 1.5hr in 1 paraffin cycle and then 30 minutes in the next paraffin cycle.
5. I then place the embedded brain tissue into a mold and I am ready for slicing.


About 50% of my brains look like this:





Around the box you can see that it is lighter in color and looks as if the paraffin did not get in there. When I cut the tissue at about 5 microns it rips every time. The weird thing is that this area is exposed directly to the paraffin during the embedding process and 2hrs has to be enough time. It is not like the problem lies deep within a piece of tissue.


Do you think I should just increase the embedding time or is there something else that is causing this anomaly?

Thanks for the help
« Last Edit: April 18, 2010, 05:24:44 PM by YouDirtyRat »

Incomplete Penatrance with Paraffin Embedding
« on: April 18, 2010, 05:22:28 PM »

Offline gula

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Re: Incomplete Penatrance with Paraffin Embedding
« Reply #1 on: April 19, 2010, 10:45:47 AM »
If paraffin penetrance isn't completed the area would be wet and mushy from the clearing agent. Your sections would spread on the waterbath like a fatdrop.
Hard and brittle tissue is due to overdehydration. Maybe the fixation time is too short, and the formally central area of the brain isn't fixed well enough. After cutting the brain in halfs underfixed area  would undergo postfixation by dehydration in 70%.
I would try to wet the blocksurface with warm tapwater for 10-30 sec. and look at the change. For new specimens let the tissue after slicing for another hour in NBF.
maybe...
gula

Offline excalibur

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Re: Incomplete Penatrance with Paraffin Embedding
« Reply #2 on: April 20, 2010, 02:10:16 PM »
Ditto gula. Incomplete fixation.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline CanuckPhD

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Re: Incomplete Penatrance with Paraffin Embedding
« Reply #3 on: April 21, 2010, 03:04:23 AM »
We work with mouse brains almost on a daily basis. Our standard protocol is to perfuse mouse with PBS (25mls) followed by 25mls of 4% PFA. If we can not perfuse with PFA because we need to do lymphocyte recall assays or gene expression we stop at PBS.

We then always bisect the brain and fix in 4% PFA. If we have perfused the mouse we either do 2hrs of PFA, prior to cryoprotection or if for paraffin over night. Without PFA perfusion 3hrs prior to cryoprotection. We never have a problem with under fixation.

We then run a 7 hour programme on the VIP processor (no vacuum except paraffin baths, 3 changes).

Re: Incomplete Penatrance with Paraffin Embedding
« Reply #3 on: April 21, 2010, 03:04:23 AM »