[Histonet] IHC on paraformaldehyde-fixed

Kemlo Rogerson Kemlo.Rogerson <@t> waht.swest.nhs.uk
Mon Dec 8 06:15:07 CST 2008


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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu =
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tony =
Henwood
Sent: 07 December 2008 22:06
To: tifei <@t> foxmail.com; Reuel Cornelia; histonet; =
anh2006 <@t> med.cornell.edu; Jan Shivers
Subject: RE: RE: RE: [Histonet] IHC on paraformaldehyde-fixed

Tf,
In answer to you email:
=20
No I do not carry toxic liquid in my car.
But does the PFA powder dissolve easily? My experience is that you need =
to make the solution alkaline then heat it.
I never add methanol to my 10% neutral buffered formalin (it is buffered =
and diluted ie 10%). The risk of polymerisation of the formalin (since =
it is diluted) and formic acid formation (since it is buffered) is =
greatly reduced.
=20

Regards=20

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=20


	-----Original Message-----
	From: tf [mailto:tifei <@t> foxmail.com]=20
	Sent: Saturday, 6 December 2008 3:01 PM
	To: Reuel Cornelia; Tony Henwood; histonet; anh2006 <@t> med.cornell.edu; =
Jan Shivers
	Subject: Re: RE: RE: [Histonet] IHC on paraformaldehyde-fixed
=09
=09
	you want to carry a bottle of toxic liquid on your car? or you will =
take a box of powder that can dissolve into useful solution easily?
	=20
	You have to add methanol in 10% formalin & 4% formaldehyde, rather 4% =
paraformaldhyde....PFA is methanol free..it's very important.
	=20
	=20
	2008-12-06=20
=09
________________________________

	tf=20
=09
________________________________

	=B7=A2=BC=FE=C8=CB=A3=BA Reuel Cornelia=20
	=B7=A2=CB=CD=CA=B1=BC=E4=A3=BA 2008-12-06  00:09:36=20
	=CA=D5=BC=FE=C8=CB=A3=BA Tony Henwood; tifei <@t> foxmail.com; histonet; =
anh2006 <@t> med.cornell.edu; Jan Shivers=20
	=B3=AD=CB=CD=A3=BA=20
	=D6=F7=CC=E2=A3=BA RE: RE: [Histonet] IHC on paraformaldehyde-fixed=20
=09
=09
	I have been curious about this discussion. we used 4% paraformaldehyde
	for smaller biopsies only because it has a faster penetration to tissue
	than 10% formalin. In all my IHC that I have done. I observe that doing
	an IHC with 4% paraformaldehyde does not necessarily need  antigen
	retrieval  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.=20
	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" <AnthonyH <@t> chw.edu.au> 12/04/08 9:29 PM >>>
	tf wrote:
	=20
	"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~~~."
	=20
	I have not heard this before.
	Do you have a reference for this?
	=20
	=20
	Regards=20
	Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)=20
	Laboratory Manager & Senior Scientist=20
	Tel: 612 9845 3306=20
	Fax: 612 9845 3318=20
	the children's hospital at westmead
	Cnr Hawkesbury Road and Hainsworth Street, Westmead
	Locked Bag 4001, Westmead NSW 2145, AUSTRALIA=20
	-----Original Message-----
	From: tf [mailto:tifei <@t> foxmail.com]=20
	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=20
	the difference you observed between may due to any other
	variability, or the co-fixative you used.
	=20
	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~~~.
	=20
	=20
	=20
	=20
	2008-12-05=20
	________________________________
	tf=20
	________________________________
	=B7=A2=BC=FE=C8=CB=A3=BA Tony Henwood=20
	=B7=A2=CB=CD=BC=E4=A3=BA 2008-12-05  06:00:03=20
	=CA=D5=BC=FE=C8=CB=A3=BA anh2006 <@t> med.cornell.edu; Jan Shivers; histonet =

	=B3=AD=CB=CD=A3=BA=20
	=D6=F7=A3=BA RE: [Histonet] IHC on paraformaldehyde-fixed=20
	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=20
	Cnr Hawkesbury Road and Hainsworth Street, Westmead=20
	Locked Bag 4001, Westmead NSW 2145, AUSTRALIA=20
	-----Original Message-----
	From: anh2006 <@t> med.cornell.edu [mailto:anh2006 <@t> med.cornell.edu]=20
	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 <AnthonyH <@t> chw.edu.au>
	Date: Thu, 04 Dec 2008 09:35:09=20
	To: Jan Shivers<shive003 <@t> umn.edu>;
	histonet<histonet <@t> lists.utsouthwestern.edu>
	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?=20
	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=20
	Cnr Hawkesbury Road and Hainsworth Street, Westmead=20
	Locked Bag 4001, Westmead NSW 2145, AUSTRALIA=20
	-----Original Message-----
	From: histonet-bounces <@t> lists.utsouthwestern.edu=20
	[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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