Fwd: Re: Fwd: Re: [Histonet] mouse brains

[Atoska S. Gentry]

>Date: Thu, 07 Oct 2004 14:47:42 -0500
>To: LuAnn Anderson <ander093 <@t> tc.umn.edu>
>From: "Atoska S. Gentry" <gentras <@t> vetmed.auburn.edu>
>Subject: Re: Fwd: Re: [Histonet] mouse brains
>
>
>Please accept my apology, no offense intended. My original inquiry was the 
>maximum amount of time one can hold mouse brain in PFA.. You're exactly 
>correct in regards to the sectioning of animal tissues, but human tissues 
>sectioned better when chilled  23+ years ago when I was in that 
>discipline.  Sounds like your tissues are undergoing extreme dehydration 
>and you're having to rehydrate in order to get sections. Thanks again. Atoska
>
>At 01:22 PM 10/7/2004, you wrote:
>>I've been doing this for 20 years--it ALWAYS works. Wild type or knock 
>>out, makes no difference. You put them on ice after soaking to cool them 
>>down at that point. I do this on a daily basis and have never had 
>>problems or come across blocks that require complete cold at all times??? 
>>This does not make sense to me. Anyway, This is what works for me and I 
>>get beautiful sections---first time, every time. If you don''t want to 
>>try it that's fine. It was just a suggestion.
>>
>>
>>At 12:20 PM 10/7/04, you wrote:
>>
>>>Thanks! yes, I'm familiar with that procedure. I used it for the knock 
>>>out brains and it worked pretty good, but the wild type are requiring 
>>>complete cold. Fortunately, as mentioned through trial & error I've been 
>>>able to obtain sections. I'm just curious about avenues to avoid for 
>>>mouse brains to be sectioned beyond this point. Atoska
>>>
>>>At 11:46 AM 10/7/2004, you wrote:
>>>>Face blocks and soak face down in warm water or warm water with a drop 
>>>>if dish soap. Soak for at least 20 minutes (or longer). Place blocks on 
>>>>ice to cool. Sectioning should be easy after this. In 20 years, this 
>>>>method has worked flawlessly every time with animal brain sections.
>>>>
>>>>
>>>>At 10:26 AM 10/7/04, you wrote:
>>>>
>>>>>>Date: Thu, 07 Oct 2004 10:26:10 -0500
>>>>>>To: Gayle Callis <gcallis <@t> montana.edu>
>>>>>>From: "Atoska S. Gentry" <gentras <@t> vetmed.auburn.edu>
>>>>>>Subject: Re: [Histonet] mouse brains
>>>>>>
>>>>>>
>>>>>>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:
>>>>>>>Atoska,
>>>>>>>
>>>>>>>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
>>>>>>>>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>>>>>>
>>>>>>>Gayle Callis
>>>>>>>MT,HT,HTL(ASCP)
>>>>>>>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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