Fwd: { SPAM 1 }::Re: Fwd: Re: [Histonet] mouse brains

From:"Atoska S. Gentry"


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>Subject: { SPAM 1 }::Re: Fwd: Re: [Histonet] mouse brains
>Aha!! The 4C is your problem. Cold slows down fixation immensely. It is an 
>"old wive's tale" that tissues must be fixed in the refrigerator with PFA. 
>Try fixing some at room temp and compare with some fixed at 4C. You will 
>get much better fixation and therefore better results at room temp. Hope 
>this helps.
>At 02:56 PM 10/7/04, you wrote:
>>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
>>>X-Sender: gcallis@gemini.msu.montana.edu
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>>>Date: Thu, 07 Oct 2004 13:20:57 -0600
>>>To: "Atoska S. Gentry" 
>>>From: Gayle Callis 
>>>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
>>>>>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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