Author Topic: Fixing and processing times for adult mouse brain in paraffin  (Read 4953 times)

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Offline meghanucc

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Hi, I am trying to troubleshoot my paraffin-embedded mouse brain sections - they seem to be embedded in the block properly, but when I section, the brain looks a bit wrinkled with a kind of puckering on the slide, while the wax around it is perfectly fine. 

A few questions: ???

1) Can you overfix tissue?  Currently we fix in 4% PFA at 4 degrees for 48 hours.

2) Can the specimens be damaged by heat?  We use low-melting wax (54-57 degrees) but the histokinet is set at 65 degrees (factory setting) - is this unnecessary? 

3) Can they get dried out from too much time in EtOH gradients or xylene?

4) If I needed to store fixed brains, could I leave them in PFA, or should I use 70% EtOH? 

At the moment I am trying out different times for all the steps, but if anyone has experience with this or could recommend a protocol would be a HUGE help, and very much appreciated! 


Offline excalibur

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Re: Fixing and processing times for adult mouse brain in paraffin
« Reply #1 on: March 02, 2007, 08:24:00 AM »
1) For paraffin sections, you cannot overfix in formalin. In fact, for a mouse brain you could fix for one week at room temp to insure thorough fixation. With rabbit, I recommend 2 weeks minimum and even longer for larger species.

2) Yes, high paraffin temps can damage tissue. However, I'm not familiar with what the histokinet is. Is it a processor? If so, the temp for the paraffin should not be any higher than 60C on any equipment.

3) Yes, over dehydration is possible, especially with rodent tissue, which is leaner than human.

4) Store fixed tissue in 70% EtOH. Tissue can be stored this way for months, even years.

With all that said, I think the wrinkling you see on the slide is very typical for brain tissue. Try increasing the waterbath temp and let the sections float for a while and spread out. Avoid cutting blocks that are too cold. Brain cuts best at almost room temp.

I hope all this helps and good luck.

Paula K. Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
8901 S Santa Fe, Suite G
Oklahoma City, OK 73139
405-759-3959
www.excaliburpathology.com

Offline meghanucc

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Re: Fixing and processing times for adult mouse brain in paraffin
« Reply #2 on: March 02, 2007, 10:03:47 AM »
Thanks Excalibur! 

We have long debated in our lab about 'overfixing' and storing fixed brains so i will go with the 70% EtOH, that takes a lot of pressure off having to work out the days for taking brains and processing them. 

The "histokinet" as we call it is a Leica tissue processor with 12 automated steps.  The factory temp setting is 65 and can only be changed with a screwdriver so its inconvenient to mess with it when so many people use the machine.  But I will definitely go by your advice and push to have that lowered. 

Last night I sent some specimens through with decreased times for the EtOH gradients and today when I embedded they look a bit better, but I won't know til I section next week.  I'll make sure I increase the temp on the waterbath. 

Thanks for the help!


Re: Fixing and processing times for adult mouse brain in paraffin
« Reply #2 on: March 02, 2007, 10:03:47 AM »