Re: RE: [Histonet] Silly Question?

Sat Dec 13 07:48:03 CST 2008

No.
Once the tisse was fixed, u can never get it fresh again.

2008-12-13 

tf 

发件人： Rene J Buesa 
发送时间： 2008-12-13  05:23:06 
收件人： pruegg; Merced Leiker 
抄送： 'histonet <@t> lists.utsouthwestern.edu'; 'Pat Flannery' 
主题： RE: [Histonet] Silly Question? 

Just two things about crosslinking:
1- there is not such a thing as over crosslinkage, because when all the reaction sites have reacted, there are no reaction sites left to "overreact"; and
2- formaldehyde fixation is a reversible reaction and the tissue will lose the croslinkage even by placing them in distilled water instead of HIER (although it will take much more time).
Ren?J.
--- On Fri, 12/12/08, Merced Leiker <leiker <@t> buffalo.edu> wrote:
From: Merced Leiker <leiker <@t> buffalo.edu>
Subject: RE: [Histonet] Silly Question?
To: pruegg <@t> ihctech.net
Cc: "'histonet <@t> lists.utsouthwestern.edu'" <histonet <@t> lists.utsouthwestern.edu>, "'Pat Flannery'" <pjfnefro <@t> duke.edu>
Date: Friday, December 12, 2008, 2:03 PM
So I have a question about the cross-linking aspect of PFA...while I agree 
I need it to keep my epitope in place, is there such a thing as 
OVER-crosslinking (i.e., tissue spending TOO much time in formalin - weeks? 
months?) that would make my epitope difficult to near-impossible to 
retrieve?
Loving the formaldehyde soap-boxes histonetters get onto...
--On Friday, December 12, 2008 10:36 AM -0700 pruegg <@t> ihctech.net wrote:
>
> i have had this argument with the researchers at the University for 30
> years, somewhere back in the day they were told that commercially made
> formalin had methanol in it (it does but just a little and does not hurt
> anything in my experience) and that methanol would damage their tissue
> for IHC, so they think they must use paraformaldehyde and make it fresh
> themselves.  Since new people make it all the time it often does not get
> made up correctly and their stress over this issue is miss placed as they
> should be using something commercial for consistancy and paying more
> attention to adequate time for fixation in reg formalin.
> Another anoying myth that is difficult to combat with them is that
"we
> should limit the fixation time in aldehyde fixatives because it will
> cross link the proteins masking them for IHC", there fore i am always
> getting tissue that has not been fixed long enough (at least 24 hrs. to
> protect it from paraffin processing, because if the proteins are not
> cross linked they can be alcohol fixed and/or washed away forever), the
> people in research know about the cross linking fo aldehydes but do not
> know that cross linking of proteins is a good thing and they also do not
> know that we have advanced methods HIER or EIER for unmasking the
> proteins, but we have no way of getting a protein back that has been lost
> in processing because the sample was not adequately fixed.
>
> there i will get off my Friday soap box..................
>
> Happy Holidays to all!
>
> Patsy
>
>
>
> -------- Original Message --------
> Subject: RE: [Histonet] Silly Question?
> From: Merced Leiker <leiker <@t> buffalo.edu>
> Date: Fri, December 12, 2008 8:12 am
> To: "Edwards, R.E." <ree3 <@t> leicester.ac.uk>, 'Pat
Flannery'
> <pjfnefro <@t> duke.edu>
> Cc: "'histonet <@t> lists.utsouthwestern.edu'"
> <histonet <@t> lists.utsouthwestern.edu>
>
> In research lab situations particularly, one does not have the time or
> technique for nailing down the ways of making each of the buffers,
> reagents, and procedures work the "right" way or the most
optimum way...a
> lot of times it's students or postdocs just focused on getting their
> project done and not caring how their fixative is made as long as it
> "works" to some degree and, alas, it's up to us already
over-booked
> technicians to figure out the best way to make the PFA....and we usually
> don't have a whole day (week, or year) to spend researching the
> back-and-forth arguments, either! ;-)
>
> Merced
>
> --On Friday, December 12, 2008 2:04 PM +0000 "Edwards, R.E."
> <ree3 <@t> leicester.ac.uk> wrote:
>
>> You hit the nail on the head "That's what we always
use", fear of
>> change is a common human condition.
>>
>> -----Original Message-----
>> From: histonet-bounces <@t> lists.utsouthwestern.edu
>> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Pat
>> Flannery Sent: 11 December 2008 16:59
>> To: histonet <@t> lists.utsouthwestern.edu
>> Subject: [Histonet] Silly Question?
>>
>> Please humor me on this if it's obvious (to everyone but me): why
do
>> we use paraformaldehyde (which is so inconvenient to make up) rather
>> than buffered formalin or just diluted formaldehyde itself?
>>
>> It seems that around here, some folks prefer paraformaldehyde (either
>> 2% or 4%) and others use formalin, while some others stick to diluted
>> formaldehyde (I see all 4 on labels for specimens submitted for
>> histology). Is it mostly a matter of personal preference or where you
>> were trained (i.e. force of habit) or is there a valid reason to use
>> each solution (basically the same chemical once in solution, merely
>> buffered or not)? The only answer I've gotten when I've asked
is,
>> "That's what we always use."
>>
>> Thanks.
>>
>> -Pat Flannery (not a "real" histologist - I just play one in
the lab)
>>
>>
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>
>
>
> Merced M Leiker
> Research Technician II
> 354 BRB (pkgs) / 140 Farber Hall (letters)
> School of Medicine and Biomedical Sciences
> State University of New York at Buffalo
> 3435 Main St, Buffalo, NY 14214
> Ph: (716) 829-6033
> Fx: (716) 829-2725
>
> "Without my flaws I'm really very boring."
> - random internet blog commentator
>
>
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Merced M Leiker
Research Technician II
354 BRB (pkgs) / 140 Farber Hall (letters)
School of Medicine and Biomedical Sciences
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214
Ph: (716) 829-6033
Fx: (716) 829-2725
"Without my flaws I'm really very boring."
- random internet blog commentator
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