[Histonet] IHC and oven temperature

Sat May 2 15:55:06 CDT 2015

John your points do seem to make it seem somewhat counter-intuitive in regards to the temperatures suggested in the literature for high points for each step. Perhaps someone will be able to provide a complete theoretical basis for the differences. It seems though that there wouldn't be much of a directed point or purpose in heating slides dry at such high temperature for very long at that stage in the process. But during AR , we have moist and the eletrolytic conditions, so use of the higher temperature is applied for a more directed and specific effect that benefits us in identifying the particular epitope of interest..any thoughts? Seems like high temp acheives a goal in the second instance, but not much purpose is gained at the higher temperature in the first instance, and potentially damage to some aspects.  I'll await further information and discussion from the group. 
Thanks

Joelle Weaver MAOM, HTL (ASCP) QIHC

> From: jkiernan at uwo.ca
> To: Histonet at lists.utsouthwestern.edu; tony.henwood at health.nsw.gov.au
> Date: Thu, 30 Apr 2015 18:24:10 -0500
> Subject: Re: FW: [Histonet] IHC and oven temperature
> CC: 
> 
> The statement quoted by Tony from the Dako manual cannot be true because many antigens have to be exposed to water at 100C in order to be immunostained - antigen retrieval. Denaturation of a macromolecule by heat increases the number of exposed epitopes, which typically are short amino acid sequences that bind specifically to the Fab segments of antibody molecules. 
> 
> On the other hand, it is easy to believe that 60C would denature antibody molecules enough to damage their binding sites and impair or prevent immunostaining. According to AWP Vermeer and W Norde (2000), the Fab segments of IgG were denatured when the temperature of a solution slightly exceeded 60C. ("The Thermal Stability of Immunoglobulin: Unfolding and Aggregation of a Multi-Domain Protein" Biophysical Journal 78: 394–404.) They found that further heating denatured the Fc segment, but the changed molecules became entangled and aggregated before denaturation was complete. Microwave heating is sometimes used to accelerate immunostaining, but control of the temperature is critical. For example: ME Boon & E Marani (1991) "The major importance of temperature data in publications concerning microwave techniques" European Journal of Morphology 29: 181–183. 
> 
>  John Kiernan
> London, Canada
> = = =
> On 30/04/15, "Tony Henwood (SCHN)"  <tony.henwood at health.nsw.gov.au> wrote:
> > 
> > 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 preferred 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
> > > >
> > > >
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