Fwd: Re: [Histonet] mouse brains

Atoska S. Gentry gentras <@t> vetmed.auburn.edu
Thu Oct 7 14:56:41 CDT 2004

Thanks so much. You're exactly correct. Our in house fixation in 4%PFA is 
done strictly at 4C overnight, but we've not done mouse brains 
before.  Therefore, I had to devise a whole new processing schedule for 
them. Atoska

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>Date: Thu, 07 Oct 2004 13:20:57 -0600
>To: "Atoska S. Gentry" <gentras <@t> vetmed.auburn.edu>
>From: Gayle Callis <gcallis <@t> montana.edu>
>Subject: Re: [Histonet] mouse brains
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>For one thing, PFA is NOT stable, it tends to break down, go bad far too 
>fast. The brains should have been totally fixed on site, then transferred 
>to either 70% ethanol, even 50% alcohol after rinsing with PBS and shipped 
>that way.  RT fixation with PFA has always been a no no, guess left over 
>from EM days, but everyone I work with doing PFA fixation does the 
>fixation at 4C, then proceeds from there.  I suggest they suspend (if they 
>do immersion fixation) the brains, hang them so brains are not sitting on 
>bottom of a container to allow all sides/surfaces to have contact with 
>PFA, particularly if they do immersion fixation.  I would hope they start 
>doing perfusion followed by immersion, far better with brain and PFA.
>We would never store brains in PFA, rather do fixation and transfer to 
>alcohol for storage, then process. If we go longer than overnight in PFA, 
>the fixative is changed to fresh just because it is unstable.  We make 
>ours up in Dulbeccos PBS, and never exceed 60C during PFA solution 
>preparation.  Always far more difficult to deal with other peoples problems!!!
>Gayle Callis
>At 09:26 AM 10/7/2004, you wrote:
>>Gayle, thanks for your reply. Actually, I'm processing these samples for 
>>a professor from the Biological Science Department on main campus. And 
>>she's conducting various IHC stains for "Tau Filament Formation in 
>>Transgenic Mice Expressing P301L Tau" & neurofibrillary changes in CNS. 
>>She's working in conjunction with some Spanish Scientists. I believe the 
>>samples were shipped from Spain in PFA back in February 04'. When we 
>>receive them they are whole brains in PFA  from which coronal slices are 
>>taken, processed, and sectioned. The first few sets of brain received a 
>>few months ago sectioned much easier, but the two she brought out a 
>>couple of weeks ago are requiring a fair amount of trial & error to 
>>obtain fairly decent sections. Atoska
>>At 03:11 PM 10/6/2004, you wrote:
>>>More info please. Are you processing brain slices cut with a matrix 
>>>device or whole brain.  We do both and find the time in processing is 
>>>critical.   We just did whole mouse brains fixed for three weeks in NBF 
>>>for LFB staining and H&E and had wonderful sections but used a longer 
>>>processing schedule than for our coronal mouse brain slices.
>>>For our coronal sections, we perfuse after anesthetizing animal - first 
>>>with saline followed by PLP via heart (a morning protocol then fix for 
>>>an additonal 5 hours, slice into 3 mm thick slices with matrix device 
>>>prior to a special mouse brain processing schedule on a VIP the same 
>>>evening.  PLP has paraformaldehyde so will be similar to your PFA 
>>>fixation and are able to obtain excellent coronal sections.  I have not 
>>>noticed any differences between longer NBF nor PLP fixation on 
>>>sectioning effects.
>>>If we fix brain with PFA, we do this overnight at 4C, and if the 
>>>fixation needs to be longer, we change the PFA to fresh.   Are you 
>>>perfusing (ideal situation) then immersing samples overnight, immersion 
>>>only, at RT or 4C.
>>>What sectioning problems do you encounter?
>>>At 01:08 PM 10/6/2004, you wrote:
>>>>Hello, those of you processing & sectioning coronal sections of mouse 
>>>>brain; from you experience can prolonged fixation in 4%PFA  be the 
>>>>culprit of sectioning problems? What is the maximum fixation time 
>>>>period the brain should held in PFA before processing & sectioning? 
>>>>Thanks! Atoska
>>>>Atoska S. Gentry B.S., HT(ASCP)
>>>>Research Assistant III
>>>>Scott-Ritchey Research Center
>>>>College of Veterinary Medicine
>>>>Auburn University, AL  36849
>>>>Phone# (334)844-5579  Fax# (334)844-5850
>>>>Histonet mailing list
>>>>Histonet <@t> lists.utsouthwestern.edu
>>>Gayle Callis
>>>Research Histopathology Supervisor
>>>Veterinary Molecular Biology
>>>Montana State University - Bozeman
>>>PO Box 173610
>>>Bozeman MT 59717-3610
>>>406 994-6367 (lab with voice mail)
>>>406 994-4303 (FAX)

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