[Histonet] IHC on paraformaldehyde-fixed

[John Kiernan]

It is impossible for "4% paraformaldehyde" to penetrate tissues faster than "10% formalin" because both solutions contain the same concentrations of methylene glycol and formaldehyde if they are made up in a solution within several orders of magnitude of neutrality (pH 4 to 9). The usual phosphate buffers fix the pH of a formaldehyde fixative at 7.2 to 7.6, and the composition of the solution is almost exactly the same whether the solution is derived from high polymers (paraformaldehyde) or low polymers (formalin). 

Solutions of formaldehyde diluted from formalin contain a little methanol and sodium formate, which are not present if paraformaldehyde is used, but these extra ingredients do not influence penetration and have little or no effect on structural preservation, even at EM level, if the osmotic pressure of the fixative is right. See Freida Carson's textbook (Histotechnology, 2nd ed.) and also her important paper in Am. J. Clin. Path. 59: 365-373 (1973). 

John Kiernan
Anatomy, UWO
London, Canada
= = =

histonet , anh2006 <@t> med.cornell.edu, Jan Shivers 

> I have been curious about this discussion. we used 4% paraformaldehyde
> for smaller biopsies only because it has a faster penetration to 
> tissuethan 10% formalin. In all my IHC that I have done. I 
> observe that doing
> an IHC with 4% paraformaldehyde does not necessarily need 
> antigenretrieval in comparison to 10% formalin either it 
> will be human or
> animal tissue but this depends on how long was it fix, our 4%
> paraformaldehyde we fix smaller biopsies like nerve,muscle, skin 
> for 6
> to 12 hrs. and for formalin it is 12 to 48 hours or more. Maybe 
> you can
> comment on the effect on this to tissue if you say you will use 4%
> paraformaldehyde for storage and transportation. 
> 
> 
> 
> 
> Reuel Cornelia, BS MT, AMT
> Cellular Pathology
> Texas Scottish Rite Hospital for Children
> 2222 Welborn Street
> Dallas, TX 75219
> Tel: 214-559-7766
> fax: 214-559-7768
> 
> >>> "Tony Henwood" 12/04/08 9:29 PM >>>
> tf wrote:
> 
> "I DO believe that one reason some people use 4% PFA rather 10%
> formalin is that PFA is a bit more stable, both for storage and
> transportation~~~."
> 
> I have not heard this before.
> Do you have a reference for this?
> 
> 
> 
> Regards 
> 
> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) 
> Laboratory Manager & Senior Scientist 
> Tel: 612 9845 3306 
> Fax: 612 9845 3318 
> the children's hospital at westmead
> Cnr Hawkesbury Road and Hainsworth Street, Westmead
> Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 
> 
> 
> -----Original Message-----
> From: tf [mailto:tifei <@t> foxmail.com] 
> Sent: Friday, 5 December 2008 2:11 PM
> To: Tony Henwood; anh2006 <@t> med.cornell.edu; Jan Shivers;
> histonet
> Subject: Re: RE: [Histonet] IHC on paraformaldehyde-fixed
> 
> 
> the basic principles are the same for most cross-linking
> fixatives and induce similar bonds 
> the difference you observed between may due to any other
> variability, or the co-fixative you used.
> 
> I DO believe that one reason some people use 4% PFA rather 10%
> formalin is that PFA is a bit more stable, both for storage and
> transportation~~~.
> 
> 
> 
> 
> 2008-12-05 
> 
> ________________________________
> 
> tf 
> 
> ________________________________
> 
> 发件人： Tony Henwood 
> 发送间： 2008-12-05 06:00:03 
> 收件人： anh2006 <@t> med.cornell.edu; Jan Shivers; histonet 
> 抄送： 
> 主： RE: [Histonet] IHC on paraformaldehyde-fixed 
> 
> 
> Interesting point.
> Since 10% buffered formalin (made from the concentrated 38%
> formaldehyde) contain about 1% methanol, has it been shown that
> this has
> a deleterious effect on ANY antigens or are we expecting this
> worse case
> senario as being the norm?
> I am not aware of any antigens (or antigen-antibody combination)
> that
> has been badly effected by 10% formalin that is NOT effected by
> 10%
> formaldehyde. Are you aware of any??
> Regards
> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
> Laboratory Manager & Senior Scientist
> Tel: 612 9845 3306
> Fax: 612 9845 3318
> the children's hospital at westmead 
> Cnr Hawkesbury Road and Hainsworth Street, Westmead 
> Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 
> -----Original Message-----
> From: anh2006 <@t> med.cornell.edu [mailto:anh2006 <@t> med.cornell.edu] 
> Sent: Friday, 5 December 2008 1:31 AM
> To: Tony Henwood; Jan Shivers; histonet
> Subject: Re: [Histonet] IHC on paraformaldehyde-fixed
> So true. However, be aware that 10% neutral buffered formalin we
> use has
> methanol in it which may affect certain antigens so there may be
> some
> difference in staining (hence why for mouse work we now only use
> 4% PFA
> in pure PBS). It is good to be aware of the other ingredients in
> your
> fixative solutions, whether commercially prepared or a homemaede
> recipe,
> as it isn't only the formaldehyde fixative which can make a
> difference.
> -----Original Message-----
> From: Tony Henwood 
> Date: Thu, 04 Dec 2008 09:35:09 
> To: Jan Shivers;
> histonet
> Subject: RE: [Histonet] IHC on paraformaldehyde-fixed
> Gee I hate the term paraformaldehyde (as many of you probably
> know)
> This is an example of how confusion of terms can cause
> unnecessary work.
> Is "4% paraformaldehyde" different from 4 % formaldehyde?
> No
> Should any procedure done to tissues fixed in "4%
> paraformaldehyde" give
> results different to those fixed in 4% formaldehyde or 10%
> formalin? 
> No since they are the same thing.
> As Manoonkitiwongsa and Schultz (Histochem J 34: 365-367, 2002)
> state
> when paraformaldehyde actually becomes a fixative, it is no
> longer
> paraformaldehyde by chemistry or fixation capacity. Rather, it
> is
> formaldehyde in water without methanol or any other stabiliser.
> Without
> heat and an alkaline environment, paraformaldehyde in water is
> simply a
> paraformaldehyde suspension with little fixation capacity. If
> the
> fixative is prepared from paraformaldehyde then it should be
> termed 4%
> formaldehyde freshly prepared from paraformaldehyde. If a
> concentrated
> formalin solution (40% formaldehyde) is used, then it should be
> termed
> 10% formalin.
> If you do a search on Histonet for paraformaldehye, you will
> find that
> this topic has been extensively discussed.
> Regards
> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
> Laboratory
> Manager & Senior Scientist
> Tel: 612 9845 3306
> Fax: 612 9845 3318
> the children's hospital at westmead 
> Cnr Hawkesbury Road and Hainsworth Street, Westmead 
> Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu 
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
> Jan
> Shivers
> Sent: Thursday, 4 December 2008 8:34 AM
> To: histonet
> Subject: [Histonet] IHC on paraformaldehyde-fixed
> Has anyone ever done IHC on parafomaldehyde-fixed tissues, and
> if so,
> how well did it work? Will the same antigen-retrieval methods
> used with
> formalin-fixed tissue be applicable?
> I'm asking for an investigator, who already has his tissues
> fixed in
> paraformaldehyde.
> Jan Shivers
> Senior Scientist
> Pathology Teaching Program
> Histology/IHC/EM Section Head
> University of Minnesota
> Veterinary Diagnostic Laboratory
> 1333 Gortner Ave.
> St. Paul, MN 55108
> 612-624-7297
> shive003 <@t> umn.edu_______________________________________________
> 
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