Re: [Histonet] IHC and oven temperature
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: jkier...@uwo.ca
> To: Histonet@lists.utsouthwestern.edu; tony.henw...@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)" 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 [joellewea...@hotmail.com]
> > Sent: Saturday, 25 April 2015 5:51 AM
> > To: Tony Henwood (SCHN); WILLIAM DESALVO; Preiszner, Johanna
> > Cc: histonet@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.henw...@health.nsw.gov.au
> > > To: wdesalvo@outlook.com; preis...@mail.etsu.edu
> > > Date: Fri, 24 Apr 2015 09:43:59 +
> > > Subject: RE: [Histonet] IHC and oven temperature
> > > CC: histonet@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
> &g
Re: [Histonet] IHC and oven temperature
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.scou...@leicabiosystems.com] Sent: Friday, 1 May 2015 7:51 AM To: Tony Henwood (SCHN); Histonet@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.scou...@leicabiosystems.com http://www.myneurolab.com Ph. 630 964 0501 Cell 314 724 5920 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tony Henwood (SCHN) Sent: Thursday, April 30, 2015 2:32 PM To: Histonet@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 [joellewea...@hotmail.com] Sent: Saturday, 25 April 2015 5:51 AM To: Tony Henwood (SCHN); WILLIAM DESALVO; Preiszner, Johanna Cc: histonet@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.henw...@health.nsw.gov.au > To: wdesalvo@outlook.com; preis...@mail.etsu.edu > Date: Fri, 24 Apr 2015 09:43:59 + > Subject: RE: [Histonet] IHC and oven temperature > CC: histonet@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
Re: [Histonet] IHC and oven temperature
Hi Rene, Contradiction? Possibly but HIER is done in an aqueous environment, whereas the oven heating above 60oC was done dry (I will send you a copy of the article under a separate email). Regards, Tony (from down under, currently in London UK). From: Rene J Buesa [rjbu...@yahoo.com] Sent: Friday, 1 May 2015 5:56 AM To: Tony Henwood (SCHN); Histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] IHC and oven temperature What about HIER at 95-98ºC? "Everybody" uses it to "enhance" epitopes detection. Is there not an intrinsic contradiction here? René J. On Thursday, April 30, 2015 3:32 PM, Tony Henwood (SCHN) 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 [joellewea...@hotmail.com<mailto:joellewea...@hotmail.com>] Sent: Saturday, 25 April 2015 5:51 AM To: Tony Henwood (SCHN); WILLIAM DESALVO; Preiszner, Johanna Cc: histonet@lists.utsouthwestern.edu<mailto:histonet@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.henw...@health.nsw.gov.au<mailto:tony.henw...@health.nsw.gov.au> > To: wdesalvo@outlook.com<mailto:wdesalvo@outlook.com>; > preis...@mail.etsu.edu<mailto:preis...@mail.etsu.edu> > Date: Fri, 24 Apr 2015 09:43:59 + > Subject: RE: [Histonet] IHC and oven temperature > CC: > histonet@lists.utsouthwestern.edu<mailto:histonet@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-boun...@lists.utsouthwestern.edu<mailto:histonet-boun...@lists.utsouthwestern.edu> > > [histonet-boun...@lists.utsouthwestern.edu<mailto:histonet-boun...@lists.utsouthwestern.edu>] > on behalf of WILLIAM DESALVO > [wdesalvo@outlook.com<mailto:wdesalvo....@outlook.com>] > Sent: Tuesday, 21 April 2015 1:56 AM > To: Preiszner, Johanna > Cc: > histonet@lists.utsouthwestern.edu<mailto:histonet@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 > > mailto:preis...@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 > > > > > > _
Re: [Histonet] IHC and oven temperature
What about HIER at 95-98ºC? "Everybody" uses it to "enhance" epitopes detection.Is there not an intrinsic contradiction here? René J. On Thursday, April 30, 2015 3:32 PM, Tony Henwood (SCHN) 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 [joellewea...@hotmail.com] Sent: Saturday, 25 April 2015 5:51 AM To: Tony Henwood (SCHN); WILLIAM DESALVO; Preiszner, Johanna Cc: histonet@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.henw...@health.nsw.gov.au > To: wdesalvo@outlook.com; preis...@mail.etsu.edu > Date: Fri, 24 Apr 2015 09:43:59 + > Subject: RE: [Histonet] IHC and oven temperature > CC: histonet@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-boun...@lists.utsouthwestern.edu > [histonet-boun...@lists.utsouthwestern.edu] on behalf of WILLIAM DESALVO > [wdesalvo@outlook.com] > Sent: Tuesday, 21 April 2015 1:56 AM > To: Preiszner, Johanna > Cc: histonet@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 > > 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@lists.utsouthwestern.edu > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > * > This email and any files transmitted with it are confidential and intended > solely for the use of the individual or entity to whom they are addressed. If > you are not the intended recipient, please delete it and notify the sender. > > Views expressed in this message and any attachments are those of the > individual sender, and are not necessarily the views of The Sydney Children's > Hospitals Network. > > This note also confirms that this email message has been virus scanned and > although no
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.henw...@health.nsw.gov.au > To: wdesalvo@outlook.com; preis...@mail.etsu.edu > Date: Fri, 24 Apr 2015 09:43:59 +0000 > Subject: RE: [Histonet] IHC and oven temperature > CC: histonet@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-boun...@lists.utsouthwestern.edu > [histonet-boun...@lists.utsouthwestern.edu] on behalf of WILLIAM DESALVO > [wdesalvo@outlook.com] > Sent: Tuesday, 21 April 2015 1:56 AM > To: Preiszner, Johanna > Cc: histonet@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 > > 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@lists.utsouthwestern.edu > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > * > This email and any files transmitted with it are confidential and intended > solely for the use of the individual or entity to whom they are addressed. If > you are not the intended recipient, please delete it and notify the sender. > > Views expressed in this message and any attachments are those of the > individual sender, and are not necessarily the views of The Sydney Children's > Hospitals Network. > > This note also confirms that this email message has been virus scanned and > although no computer viruses were detected, The Sydney Childrens Hospital's > Network accepts no liability for any consequential damage resulting from > email containing computer viruses. > * > > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC and oven temperature
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-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of WILLIAM DESALVO [wdesalvo@outlook.com] Sent: Tuesday, 21 April 2015 1:56 AM To: Preiszner, Johanna Cc: histonet@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 > 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@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Sydney Children's Hospitals Network. This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Sydney Childrens Hospital's Network accepts no liability for any consequential damage resulting from email containing computer viruses. * ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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 > 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@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
