[Histonet] fixation times

Bryan Hewlett bhewlett <@t> cogeco.ca
Tue Mar 4 12:21:27 CST 2008 

Melissa,

What type of section- cryo or paraffin?

I hate to tell you this, but after more than 40 years of IHC experience with 
both animal and human tissues,
I have yet to find an antibody for paraffin sections that does not work more 
consistently after a minimum of 24 hours fixation in formaldehyde.
After only 4-6 hours formaldehyde fixation your paraffin tissues are 
essentially alcohol fixed in the processor!
For some epitopes e.g. vimentin, cytokeratins  and other intermediate 
filaments, that's fine, but for most cell surface proteins NOT.


Bryan


----- Original Message ----- 
From: "Melissa Mazan" <melissa.mazan <@t> tufts.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Tuesday, March 04, 2008 1:10 PM
Subject: [Histonet] fixation times


> This thread has been very interesting - and I have a practical question. 
> We do IHC - both immunofluorescent and immunoenzyme - on lung tissue in 
> our lab.  We generally try to fix the tissues in formalin for at least 4 
> hours and no more than 6 hours - as we tend to get better antibody 
> binding - this is even with HIER.  Is there an optimum amount of time that 
> the group recommends? Thanks - Melissa
>
> histonet-request <@t> lists.utsouthwestern.edu wrote:
>
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>> Today's Topics:
>>
>>    1. RE: Washing out formalin fixation (Lengthy)
>>       (Marshall Terry Dr, Consultant Histopathologist)
>>    2. What happened to BIOMEDA <pkarlisch <@t> psu.edu> (Patricia Karlisch)
>>    3. Re: Histonet Digest, Vol 52, Issue 4 (MKing)
>>    4. Re: Washing out formalin fixation (Lengthy) (Bryan Hewlett)
>>
>>
>> ----------------------------------------------------------------------
>>
>> Message: 1
>> Date: Tue, 4 Mar 2008 17:35:04 -0000
>> From: "Marshall Terry Dr, Consultant Histopathologist"
>> <Terry.Marshall <@t> rothgen.nhs.uk>
>> Subject: RE: [Histonet] Washing out formalin fixation (Lengthy)
>> To: "Morken, Tim" <tim.morken <@t> thermofisher.com>,
>> <histonet <@t> lists.utsouthwestern.edu>
>> Message-ID:
>> <5C0BED61F529364E86309CADEA63FEF20163F358 <@t> TRFT-EX01.xRothGen.nhs.uk>
>> Content-Type: text/plain; charset="us-ascii"
>>
>>  "These studies these show that fixation of peptide spots (essentially
>> zero thickness) attached to glass slides take over 6 hours to "fix" at
>> room temperature. In this case they defined "fixed" as the point at
>> which an antibody  would no longer detect it's target epitope, or the
>> reaction was severely diminished. They also showed that HIER reverses
>> the "fixation"
>>
>> So far so good, and supports what I have been saying in this forum for
>> years about the (long) time it takes to formalin fix tissue, and the
>> misbelief that wetting them for a few hours is fixing.
>> But then......
>>
>> "This calls into question any method that relies on less than six hours
>> formalin fixation at room temperature (ie, biopsies, just because they
>> are small) and also the effect of exposing those short-fixed tissues to
>> long exposure to 70% alcohol, or other aqueous solution, before clearing
>> and embedding."
>>
>> I know not what this all means/is getting at.
>>
>> Terry
>>
>> -----Original Message-----
>> From: histonet-bounces <@t> lists.utsouthwestern.edu
>> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Morken,
>> Tim
>> Sent: 04 March 2008 17:17
>> To: histonet <@t> lists.utsouthwestern.edu
>> Subject: RE: [Histonet] Washing out formalin fixation (Lengthy)
>>
>>
>> Thanks for the synopsis Bryan.
>>
>> I'd like to point out that the 2004 paper by Sompuram,and a followup in
>> 2006 are very interesting. These studies these show that fixation of
>> peptide spots (essentially zero thickness) attached to glass slides take
>> over 6 hours to "fix" at room temperature. In this case they defined
>> "fixed" as the point at which an antibody  would no longer detect it's
>> target epitope, or the reaction was severly diminished. They also showed
>> that HIER reverses the "fixation."
>>
>>
>> The 2006 paper also investigates why some epitopes are affected by
>> formalin and others are not.
>>
>> Sompuram SR, Vani K, Messana E and Bogen SA. A molecular mechanism of
>> formalin fixation and antigen retrieval. Am J Clin Pathol (2004);121:
>> 190-199
>>
>> Sompuram, et. al, A molecular model of antigen retrieval using a peptide
>> array, Am J Clin Path 2006;125:91-98
>>
>>
>> Tim Morken
>> Technical Support Manager
>> Lab Vision Products
>> Anatomical Pathology
>> ThermoFisher Scientific
>>
>> -----Original Message-----
>> From: histonet-bounces <@t> lists.utsouthwestern.edu
>> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Bryan
>> Hewlett
>> Sent: Tuesday, March 04, 2008 7:56 AM
>> To: Johnson, Teri; histonet <@t> lists.utsouthwestern.edu
>> Subject: Re: [Histonet] Washing out formalin fixation (Lengthy)
>>
>> Hi Teri and everyone else on this thread,
>>
>> Washing out many of the effects of formaldehyde fixation on tissues with
>> running water has been known for years (much longer than this old boy
>> has been around).
>> In modern terms, it is the essential underlying mechanism for so-called
>> antigen retrieval (HIER).
>>
>> Formaldehyde fixes proteins by addition, with the formation of
>> hydroxymethyl adducts on the reactive side chains of proteins.
>> Once enough of these hydroxymethyl adducts are formed, and IF they are
>> in close approximation to each other, they may slowly cross-link by the
>> formation of methylene bridges.
>> However, these adducts and initial cross-links are unstable and readily
>> reversed by water and alcohol (see Kiernan (1999).
>> It takes 24 hours at room temperature for all the hydroxymethyl adducts
>> to form, i.e. maximal binding threshold (see Fox et al, 1985).
>> If the tissue is then exposed to running water before all the adducts
>> have formed (i.e. less than 24 hours), the reversal is very rapid.
>> The shorter the time in formaldehyde, the more rapid the reversal.
>> Even after the essential 24 hours fixation and also after a more lengthy
>> 6 days fixation, running water will still remove the adducts and
>> hydrolyse the methylene bridges.
>> There is at least one publication (Helander, 1994) that provides data
>> regarding this effect.
>> After 24 hours fixation, 50% reversal occurred in less than 24 hours,
>> 90% reversal was obtained after 6 days washing and for 6 days fixation
>> 90% reversal after 4 weeks washing.
>> It should be noted that these reversal times were obtained at ambient
>> temperatures and the times may be considerably reduced by elevated
>> temperatures.
>>
>> This reversal effect is also obtained on tissue sections that have been
>> processed to wax.
>> However, because of the additional shrinkage and hydrophobicity of the
>> processed proteins, the reversal is slowed somewhat until the proteins
>> re-hydrate.
>> The reversal effect can also be aided by the presence of other ions in
>> the water (the purpose of HIER buffers).
>> Back in the sixties, in order to successfully demonstrate Ig's by IF, we
>> were reversing the fixation effects on paraffin sections by placing them
>> in hypotonic buffers for 2 days at 37C.
>> Today, since we are all in a great rush for results, we obviously drive
>> the reversal at higher temperatures to speed things up!!
>>
>> References:
>>
>> Baker JR (1958), Principles of Biological Microtechnique, Methuen & Co.
>> Ltd.
>> Hopwood D. Fixatives and fixation: A review. Histochemical journal
>> (1969); 1, p19-55 Burnett MG. The mechanism of the formaldehyde clock
>> reaction: Methylene glycol dehydration. J Chem. educ. (1982); 59, 160
>> Fox CH et al. Formaldehyde fixation. J Histochem Cytochem (1985); 33,
>> 845-853
>> Helander KG. Kinetic studies of formaldehyde binding in tissue.
>> Biotechnique and Histochemistry. (1994); 69, 177-179 Kiernan J.A.,
>> Histological and Histochemical Methods: Theory and Practice, 3rd
>> Edition, (1999). Oxford: Butterworth-Heinemann. ISBN # 0-7506-3106-6.
>> Shi SR, Gu J. and Taylor CR. Antigen Retrieval Techniques:
>> Immunohistochemistry and Molecular Morphology. (2000) Eaton Publishing.
>> ISBN 1-881299-43-0.
>> Sompuram SR, Vani K, Messana E and Bogen SA. A molecular mechanism of
>> formalin fixation and antigen retrieval. Am J Clin Pathol (2004);121:
>> 190-199
>>
>>
>> Best regards,
>>
>> Bryan
>>
>> ----- Original Message -----
>> From: "Johnson, Teri" <TJJ <@t> Stowers-Institute.org>
>> To: <histonet <@t> lists.utsouthwestern.edu>
>> Sent: Monday, March 03, 2008 2:33 PM
>> Subject: [Histonet] Washing out formalin fixation
>>
>>
>> Last week, a researcher here asked me what the chemical mechanism was of
>>
>> washing out the effects of formalin fixation on the tissues with running
>>
>> water. In other words, how does it work? Anybody here know?
>>
>> Teri Johnson, HT(ASCP)QIHC
>> Managing Director Histology Facility
>> Stowers Institute for Medical Research
>> 1000 E. 50th St.
>> Kansas City, MO 64110
>>
>>
>> _______________________________________________
>> Histonet mailing list
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>>
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>>
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>> ------------------------------
>>
>> Message: 2
>> Date: Tue, 04 Mar 2008 12:43:42 -0500
>> From: "Patricia Karlisch" <pkarlisch <@t> hmc.psu.edu>
>> Subject: [Histonet] What happened to BIOMEDA <pkarlisch <@t> psu.edu>
>> To: <Histonet <@t> lists.utsouthwestern.edu>
>> Message-ID: <47CD43FD.07B7.008C.0 <@t> hmc.psu.edu>
>> Content-Type: text/plain; charset=US-ASCII
>>
>> Histonetters,
>>      I can not contact Biomeda any longer. Does anyone know what 
>> happened?   Thanks, Pat
>>  Pat Karlisch
>> Supervisor, Histology, Pathology and Laboratory Medicine
>> Penn State Milton S. Hershey Medical Center
>> Mail Code H179
>> Hershey, PA 17033
>> Phone (717) 531-6072
>> Fax: (717) 531- 7741
>> email: pkarlisch <@t> psu.edu *****E-Mail Confidentiality Notice*****
>> This message (including any attachments) contains information intended 
>> for a specific individual(s) and purpose that may be privileged, 
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>> to the sender indicating this error and delete the transmission from your 
>> system immediately.
>>
>>
>> ------------------------------
>>
>> Message: 3
>> Date: Tue, 04 Mar 2008 12:48:16 -0500
>> From: MKing <making <@t> ufl.edu>
>> Subject: [Histonet] Re: Histonet Digest, Vol 52, Issue 4
>> To: histonet <@t> lists.utsouthwestern.edu
>> Message-ID: <47CD8B60.3030101 <@t> ufl.edu>
>> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>>
>> Bryan and Tim,
>> Thanks for excellent postsMK, this info will go in my tech folder!  It 
>> looked like this thread was headed into a flame war, nice to have 
>> substantiated data prevail here.
>> Mike King
>> UF Pharmacology & Therapeutics
>> --------------------
>> Date: Tue, 4 Mar 2008 10:56:18 -0500
>> From: "Bryan Hewlett" <bhewlett <@t> cogeco.ca>
>> Subject: Re: [Histonet] Washing out formalin fixation (Lengthy)
>>
>>
>>
>>
>> ------------------------------
>>
>> Message: 4
>> Date: Tue, 4 Mar 2008 12:55:19 -0500
>> From: "Bryan Hewlett" <bhewlett <@t> cogeco.ca>
>> Subject: Re: [Histonet] Washing out formalin fixation (Lengthy)
>> To: <histonet <@t> lists.utsouthwestern.edu>, "Morken, Tim"
>> <tim.morken <@t> thermofisher.com>
>> Message-ID: <002401c87e20$e90325d0$6500a8c0 <@t> mainbox>
>> Content-Type: text/plain; format=flowed; charset="iso-8859-1";
>> reply-type=original
>>
>> Hi Tim,
>>
>> Thanks, I am aware of both papers and also a follow-up discussion at BSC 
>> last year.
>> The authors also indicate that some epitopes do not mask or require HIER 
>> to demonstrate, even though they have been 'fixed' for long enough.
>> This is of course born out by every day experience in the IHC lab.
>> Interestingly, they also point out that some peptides that are fixed this 
>> way and do not require HIER, WILL require HIER if fixed in the presence 
>> of another protein or peptide.
>> That of course is also the case in the real world of tissues!!!
>>
>> (See also Yang K, Sompuram SR, Fitzgibbons P Bogen SA. National HER2 
>> proficiency test results using standardized quantitative controls: 
>> characterization of laboratory failures.
>> Arch Pathol Lab Med 112, February 2008 pp 211-216.)
>>
>> As you know, I have long decried the practice of short fixing small 
>> biopsies and the effects of reversal and refixation by alcohol during 
>> processing.
>> This can be particularly devastating on surface proteins such as HER2 
>> etc. etc.
>> The 6 hour minimum fixation time in the HER2 guidelines is, in my 
>> opinion, wrong!!!
>> OK, I'll step off the soap box.
>>
>>
>> cheers,
>>
>> Bryan
>>
>>
>>
>>
>>
>>
>>
>> ----- Original Message ----- 
>> From: "Morken, Tim" <tim.morken <@t> thermofisher.com>
>> To: <histonet <@t> lists.utsouthwestern.edu>
>> Sent: Tuesday, March 04, 2008 12:16 PM
>> Subject: RE: [Histonet] Washing out formalin fixation (Lengthy)
>>
>>
>>
>> Thanks for the synopsis Bryan.
>>
>> I'd like to point out that the 2004 paper by Sompuram,and a followup in
>> 2006 are very interesting. These studies these show that fixation of
>> peptide spots (essentially zero thickness) attached to glass slides take
>> over 6 hours to "fix" at room temperature. In this case they defined
>> "fixed" as the point at which an antibody  would no longer detect it's
>> target epitope, or the reaction was severly diminished. They also showed
>> that HIER reverses the "fixation." This calls into question any method
>> that relies on less than six hours formalin fixation at room temperature
>> (ie, biospies, just because they are small) and also the effect of
>> exposing those short-fixed tissues to  long exposure to 70% alcohol, or
>> other aqueous solution, before clearing and embedding.
>>
>> The 2006 paper also investigates why some epitopes are affected by
>> formalin and others are not.
>>
>> Sompuram SR, Vani K, Messana E and Bogen SA. A molecular mechanism of
>> formalin fixation and antigen retrieval. Am J Clin Pathol (2004);121:
>> 190-199
>>
>> Sompuram, et. al, A molecular model of antigen retrieval using a peptide
>> array, Am J Clin Path 2006;125:91-98
>>
>>
>> Tim Morken
>> Technical Support Manager
>> Lab Vision Products
>> Anatomical Pathology
>> ThermoFisher Scientific
>>
>> -----Original Message-----
>> From: histonet-bounces <@t> lists.utsouthwestern.edu
>> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Bryan
>> Hewlett
>> Sent: Tuesday, March 04, 2008 7:56 AM
>> To: Johnson, Teri; histonet <@t> lists.utsouthwestern.edu
>> Subject: Re: [Histonet] Washing out formalin fixation (Lengthy)
>>
>> Hi Teri and everyone else on this thread,
>>
>> Washing out many of the effects of formaldehyde fixation on tissues with
>> running water has been known for years (much longer than this old boy
>> has been around).
>> In modern terms, it is the essential underlying mechanism for so-called
>> antigen retrieval (HIER).
>>
>> Formaldehyde fixes proteins by addition, with the formation of
>> hydroxymethyl adducts on the reactive side chains of proteins.
>> Once enough of these hydroxymethyl adducts are formed, and IF they are
>> in close approximation to each other, they may slowly cross-link by the
>> formation of methylene bridges.
>> However, these adducts and initial cross-links are unstable and readily
>> reversed by water and alcohol (see Kiernan (1999).
>> It takes 24 hours at room temperature for all the hydroxymethyl adducts
>> to form, i.e. maximal binding threshold (see Fox et al, 1985).
>> If the tissue is then exposed to running water before all the adducts
>> have formed (i.e. less than 24 hours), the reversal is very rapid.
>> The shorter the time in formaldehyde, the more rapid the reversal.
>> Even after the essential 24 hours fixation and also after a more lengthy
>> 6 days fixation, running water will still remove the adducts and
>> hydrolyse the methylene bridges.
>> There is at least one publication (Helander, 1994) that provides data
>> regarding this effect.
>> After 24 hours fixation, 50% reversal occurred in less than 24 hours,
>> 90% reversal was obtained after 6 days washing and for 6 days fixation
>> 90% reversal after 4 weeks washing.
>> It should be noted that these reversal times were obtained at ambient
>> temperatures and the times may be considerably reduced by elevated
>> temperatures.
>>
>> This reversal effect is also obtained on tissue sections that have been
>> processed to wax.
>> However, because of the additional shrinkage and hydrophobicity of the
>> processed proteins, the reversal is slowed somewhat until the proteins
>> re-hydrate.
>> The reversal effect can also be aided by the presence of other ions in
>> the water (the purpose of HIER buffers).
>> Back in the sixties, in order to successfully demonstrate Ig's by IF, we
>> were reversing the fixation effects on paraffin sections by placing them
>> in hypotonic buffers for 2 days at 37C.
>> Today, since we are all in a great rush for results, we obviously drive
>> the reversal at higher temperatures to speed things up!!
>>
>> References:
>>
>> Baker JR (1958), Principles of Biological Microtechnique, Methuen & Co.
>> Ltd.
>> Hopwood D. Fixatives and fixation: A review. Histochemical journal
>> (1969); 1, p19-55 Burnett MG. The mechanism of the formaldehyde clock
>> reaction: Methylene glycol dehydration. J Chem. educ. (1982); 59, 160
>> Fox CH et al. Formaldehyde fixation. J Histochem Cytochem (1985); 33,
>> 845-853
>> Helander KG. Kinetic studies of formaldehyde binding in tissue.
>> Biotechnique and Histochemistry. (1994); 69, 177-179 Kiernan J.A.,
>> Histological and Histochemical Methods: Theory and Practice, 3rd
>> Edition, (1999). Oxford: Butterworth-Heinemann. ISBN # 0-7506-3106-6.
>> Shi SR, Gu J. and Taylor CR. Antigen Retrieval Techniques:
>> Immunohistochemistry and Molecular Morphology. (2000) Eaton Publishing.
>> ISBN 1-881299-43-0.
>> Sompuram SR, Vani K, Messana E and Bogen SA. A molecular mechanism of
>> formalin fixation and antigen retrieval. Am J Clin Pathol (2004);121:
>> 190-199
>>
>>
>> Best regards,
>>
>> Bryan
>>
>> ----- Original Message -----
>> From: "Johnson, Teri" <TJJ <@t> Stowers-Institute.org>
>> To: <histonet <@t> lists.utsouthwestern.edu>
>> Sent: Monday, March 03, 2008 2:33 PM
>> Subject: [Histonet] Washing out formalin fixation
>>
>>
>> Last week, a researcher here asked me what the chemical mechanism was of
>>
>> washing out the effects of formalin fixation on the tissues with running
>>
>> water. In other words, how does it work? Anybody here know?
>>
>> Teri Johnson, HT(ASCP)QIHC
>> Managing Director Histology Facility
>> Stowers Institute for Medical Research
>> 1000 E. 50th St.
>> Kansas City, MO 64110
>>
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>>
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>>
>>
>>
>>
>> ------------------------------
>>
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>> End of Histonet Digest, Vol 52, Issue 5
>> ***************************************
>
> -- 
> Melissa R. Mazan, DVM, Diplomate ACVIM
> Associate Professor and Director of Equine Sports Medicine
> Tufts Cummings School of Veterinary Medicine
> 200 Westboro Road
> North Grafton, MA 01536
> Tel:508-839-5395
> Email: melissa.mazan <@t> tufts.edu
> Fax:508-839-7922
>
>
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