Re: RE: Re: [Histonet] 70% alcohol fixation of brain

[TF]

I did try the frozen section from fresh tissue...and fix them in cold acetone for 10 min, transfer to cold PBS for 30 min, then wash in PBS at RT, begin IHC.

But the tissue quality is really tooooooooo bad. You always saw pieces of tissues floating, fall in to small pieces and gone when you washing the slide...
Under the microscope...the background is high, and you can only see granule-like existences filled by small cavities.
Actually I frozed the OCT-embedded tissue with liquid nitrogen..dont know why the tissue quality is still so bad!


2009-05-17 



TF 



发件人： Patsy Ruegg 
发送时间： 2009-05-17  01:19:09 
收件人： tifei <@t> foxmail.com; 'Geoff McAuliffe' 
抄送： Histonet <@t> lists.utsouthwestern.edu 
主题： RE: Re: [Histonet] 70% alcohol fixation of brain 
 
U would be better off freezing the brain unfixed than trying to fix in alcohol and freeze it, alcohol fixation is not recommended for freezing tissue.  Just freeze slices of the brain or a whole rat or mouse brain and then make sections and fix the sections in alcohol/acetone mixture.  Sectioning is not easy but this is a better approach than fixing in alcohol and then trying to freeze.  I would try to get some frozen sections from the unfixed frozen brain for use with antibodies not formalin friendly and then thaw fix the frozen block in formalin for several day, infiltrate it in 30% sucrose and refreeze, you should be able to get better sections then, but the tissue has to be fixed before you can cryoprotect in sucrose.
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 215
Aurora, CO 80010
P-720-859-4060
F-720-859-4110
email pruegg <@t> ihctech.net
website www.ihctech.net
IHC Resource Group www.ihcrg.org 
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of TF
Sent: Friday, May 15, 2009 12:15 PM
To: Geoff McAuliffe
Cc: Histonet <@t> lists.utsouthwestern.edu
Subject: Re: Re: [Histonet] 70% alcohol fixation of brain
thanks to the reply.
some antigens will be damaged by PFA fixation and can not be retreieval.
the shock frozen section with acetone is of quite bad tissue quality. therefore I am trying alcohol!
2009-05-16 
TF 
发件人： Geoff McAuliffe 
发送时间： 2009-05-15  23:55:15 
收件人： tifei 
抄送： Histonet <@t> lists.utsouthwestern.edu 
主题： Re: [Histonet] 70% alcohol fixation of brain 

Greetings TF:
Good luck freezing a brain that is full of alcohol! Have you checked the 
freezing point of alcohol?
Why are you doing this?
Immersing a whole brain in 70% alcohol, why??
30% sucrose is a cryoprotectant so the brain is not full of holes from 
ice crystals.Alcohol defeats this purpose.
Geoff
TF wrote:
> Hi, i dont want to use PFA for the brain fixation (rat).
> Now I tried to perfuse the rat with saline, followed with 70% alcohol. Then I do post-fixation at room temperature for 24 hours.
> I also tried saline perfusion, then I directly put the whole brain into 70% alcohol.
> Is this fine?
>
> Also, for dehydration before cutting frozen sections on a cryostat/microtome, should I use 95% alcohol? I am now using 30% sucrose~
>
> 2009-05-15 
>
>
>
> TF 
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> Histonet <@t> lists.utsouthwestern.edu
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Neuroscience and Cell Biology
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