[Histonet] IHC and oven temperature

Fri May 1 15:27:39 CDT 2015

Hi Charles,

"In this study, having previously heated the tissue to 80 degrees would improve and speed up antibody-antigen reaction by ensuring the antigen was denatured."

But not all antibodies, some were not affected, others were adversely affected. The cell and cell molecules are complex and variable.

I will send you a copy of the paper in a separate email

Regards
Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Laboratory Manager & Senior Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

________________________________________
From: Scouten, Charles [Charles.Scouten at LeicaBiosystems.com]
Sent: Friday, 1 May 2015 7:51 AM
To: Tony Henwood (SCHN); Histonet at lists.utsouthwestern.edu
Subject: RE: [Histonet] IHC and oven temperature

I have recently reviewed literature about sacrifice microwave fixation.  Proteins in the living brain are denatured in as little as .3 seconds, stopping all brain chemical reactions (all catalyzed by enzymes to tightly regulate brain), freezing the chemical state of the brain.  Proteins, including enzymes, in the mammalian body denature between about 40 degrees C to 80 degrees C.  Depending on the specific protein, each of which would have a denature temperature.  In formation, proteins fold, and then fold again.  Enzyme active sites are in the tertiary structure.  Proteins can be denatured, unfolded, destroying enzymes, without losing antigenicity, depending on what the antibody is attaching to.  Heating up to 90 degrees or higher does not break apart the proteins amino acid chain, which has much stronger bonds than the hydrogen bonds that hold the protein folded.

My guess about the statement below is that it oversimplifies.  Depending on which antigen you are going for, what its own denature temperature is, and whether the antibody in question reaches across folds to bind (is this known in any case?) you may or may not lose antigenicity between 40 degrees C and 80 degrees C.

Some monoclonal antibodies raised with a native protein bind preferentially to the denatured antigen
Bertrand Friguet,
Lisa Djavadi-Ohaniance,
Michel E. Goldberg

In this study, having previously heated the tissue to 80 degrees would improve and speed up antibody-antigen reaction by ensuring the antigen was denatured.

Cordially,

Charles W. Scouten, Ph.D.
Applications Specialist
Leica Biosystems
Charles.Scouten at Leicabiosystems.com
http://www.myneurolab.com
Ph.  630 964 0501
Cell  314 724 5920

-----Original Message-----
From: histonet-bounces at lists.utsouthwestern.edu [mailto:histonet-bounces at lists.utsouthwestern.edu] On Behalf Of Tony Henwood (SCHN)
Sent: Thursday, April 30, 2015 2:32 PM
To: Histonet at lists.utsouthwestern.edu
Subject: FW: [Histonet] IHC and oven temperature

Yes,

I read the Dako IPX educational guides (5th ed) and on page 32:
"No processes should raise tissue temperature to higher than 60oC as this will cause severe loss of antigenicity that may not be recoverable"
Unfortunately there is no evidence given or cited that validates this statement. Even though this could be right (and there are several papers that have looked at this), this statement is scientifically weak and we should not cite this as truth.

Now I do recommend the Dako reference series to my students, and I have contributed to one of these texts myself (Microscopic control of routine H&E - know your histology) but I request my students to continue to question what they read and confirm the scientific validity of the information.

Regards,
Tony

________________________________________
From: Joelle Weaver [joelleweaver at hotmail.com]
Sent: Saturday, 25 April 2015 5:51 AM
To: Tony Henwood (SCHN); WILLIAM DESALVO; Preiszner, Johanna
Cc: histonet at lists.utsouthwestern.edu
Subject: RE: [Histonet] IHC and oven temperature

I remember reading that the preffered temperature was about 60 degrees Celsius. I think that this was in the Dako education guides if I'm not mistaken. If that is the case, the citation for the source is probably in that resource available as pdf from their website.

Joelle Weaver MAOM, HTL (ASCP) QIHC

> From: tony.henwood at health.nsw.gov.au
> To: wdesalvo.cac at outlook.com; PREISZNE at mail.etsu.edu
> Date: Fri, 24 Apr 2015 09:43:59 +0000
> Subject: RE: [Histonet] IHC and oven temperature
> CC: histonet at lists.utsouthwestern.edu
>
> Hi temp drying shown to be a bad idea:
>
> Henwood, A., (2005) "Effect of Slide Drying at 80°C on Immunohistochemistry" J Histotechnol 28(1):45-46.
>
> Abstract
>
> Prolonged high temperature dry heating has been found to be deleterious to the immunohistochemical demonstration of several antigens in formalin-fixed, paraffin- embedded sections. Paraffin sections were dried at 80°C for 7 h and their immunoreactivity was compared with mirror sections dried for 1 h at 60°C. NCL-5D3, CMV, S100, HMB45, and CEA were quite labile to dry overheating whereas AElAE3, HBsAg, HBcAg, HSVII, EMA, chromogranin, and NSE were found to be quite resistant. It is recommended that coated slides (poly-L-lysine or aminopropyltriethoxysilane) and low-temperature drying (<60°C) be routinely used for irnmunohistochemistry.
>
> ________________________________________
> From: histonet-bounces at lists.utsouthwestern.edu
> [histonet-bounces at lists.utsouthwestern.edu] on behalf of WILLIAM
> DESALVO [wdesalvo.cac at outlook.com]
> Sent: Tuesday, 21 April 2015 1:56 AM
> To: Preiszner, Johanna
> Cc: histonet at lists.utsouthwestern.edu
> Subject: Re: [Histonet] IHC and oven temperature
>
> Dry heat compared to wet heat. Do not "dry" your slides at high heat.
> You are removing water trapped between slide and paraffin section.
> Antigen retrieval is an entirely different process. So not try to
> combine the two processes
>
> Sent from my iPhone
>
> > On Apr 20, 2015, at 8:48 AM, Preiszner, Johanna <PREISZNE at mail.etsu.edu> wrote:
> >
> > Hi Netters,
> >
> > is there something wrong with this logic:
> >
> > "If the tissue needs 95C for HIER, it's ok to dry the slides in an 82C oven."
> >
> > Of course I'll test it before I try it on real specimens, but maybe someone else already knows the answer...
> >
> > Thanks!
> >
> > Hanna Preiszner
> > ETSU/QCOM
> >
> >
> > _______________________________________________
> > Histonet mailing list
> > Histonet at lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
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