[Histonet] fixation times

Melissa Mazan melissa.mazan <@t> tufts.edu
Tue Mar 4 12:10:04 CST 2008


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

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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
> Histonet <@t> lists.utsouthwestern.edu
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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*****
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> 
> 
> ------------------------------
> 
> 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
> 
> _______________________________________________
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> 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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