RE: [Histonet] Re: undecalcified bone IHC
I might also add that Neil Hand is co-speaking with myself and Philip Seifert this year at the annual National Society for Histotechnology - Symposium/Convention in Vancouver B.C. Our workshop is titled: Resin Applications Forum: Methods for Processing, Special Staining, Immunohistochemical and In Situ Hybridization of Soft and Hard Tissue Including Medical Device Implants During the last 50 years, numerous histological procedures have been described on resin embedded tissue. While different types of resins are available for different purposes, the acrylics provide the widest range of techniques, especially for light microscopy applications. However, as demand from HE to more sophisticated techniques increases, so too have the problems, and nowhere is this more apparent and controversial than in the application of immunohistochemistry on resin sections. This workshop will provide a review and discussion for those individuals that currently work with and/or are just getting started working with soft and hard tissue specimens and specifically the various resins (i.e. MMA, GMA, Technovit, Acrylosin, etc.) associated with their specific tissue interests. The workshop will also detail the preparation and staining of sections of soft and hard tissue, including implants (e.g. undemineralized bone and cardiovascular stents), for immunohistochemical and in situ hybridization staining using different acrylic and epoxy resin embedding media. Specific problems and pitfalls, either technical or operational associated with certain resin embedding procedures, will be illustrated and examined. Particular emphasis will be given to procedures which have been used extensively for routine diagnostic, and research purposes, i.e. those that WORK! Individuals with a current or future intent to process and cut undemineralized tissue or tissue containing foreign implant materials using acrylic or epoxy resins are strongly encouraged to attend this workshop! Please feel free to contact me if you would like more information about the workshop as information relevant to the exact date and time becomes available. All I know at this time is that the NSH meeting is September 29th - October 3rd, 2012. Best Regards, Jack Jack Ratliff Hard Tissue Histologist Chairman, Hard Tissue Committee - National Society for Histotechnology From: gayle.cal...@bresnan.net To: histonet@lists.utsouthwestern.edu Date: Mon, 12 Mar 2012 11:04:20 -0600 Subject: [Histonet] Re: undecalcified bone IHC Jeff, It is most certainly possible to do IHC on undecalcifed bone sections embedded in PMMA although not the easiest task. Sectioning is done on a microtome that is powerful enough to cut the plastic and using tungsten carbide knives. The key is total removal of the plastic from MMA embedded bone sections to allow antibody/ immunoglobulins to access antigenic sites. Neil Hand has done IHC successfully on PMMA embedded tissues including undecalcified bone on 2 to 3 µm thick sections. I think one could cut thicker sections at 4 to 5 µm and still be successful. I do not recall what Troiano et al used. The following publications will help you and should include protocols, although conventional protocols will work according to Hand. Blythe D. Hand N et al 1997 J Clin Path 50:45-49. The use of methyl methacrylate resin for embedding bone marrow trephine biopsies. Hand NM et al 1996 Antigen unmasking using microwave heating on formalin fixed tissue embedded in methyl methacrylate J Cellular Path 1:31-37 Jackson P et al. 1996 Amplification of immunocytochemical reactions by the catalytic deposition of biotin on tissue sections. J Path 170(suppl):23A. This was about tyramide amplification when one gets a weak signal from conventional methods. Hand NM, Church RJ 1998 Superheating using pressure cooking: its use and application in unmasking antigens embedded in methyl methacrylate. J Histotechnology 2`:231-236 Hand NM et al 1989 Immunohistochemistry on resin embedded tissue for light microscopy: a novel post embedding procedure. Proceeding Royal Microscopical Society 24(1):A54-55. Hand NM Plastic Embedding media and techniques, Ch.30, p 663-677. Theory and Practice of Histological Technique, 5th edition by Gamble and Bancroft. The 6th edition is updated under same title. Use Google Scholar to find Troiano N et al from Yale on doing IHC on PMMA embedded bone sections with publications in J Histotechnology. Hand mentioned several HIER methods, using citrate buffer. Optimizing retrieval will depend on the antigen and you may end up doing this with some form of HIER, including microwave or other heat producing methods and with different buffers. Enzyme digestion is also a possibility. Hand removed MMA with xylene, warm my speed up the removal, also more than one change for 10 - 20 minutes or longer. When I talked to him
Re: [Histonet] Re: undecalcified bone IHC
Excellent! Happy to read the venerable Neil Hand is coming back to the symposium, always a great speaker! -Damien On Tue, Mar 13, 2012 at 10:50 AM, Jack Ratliff ratliffj...@hotmail.comwrote: I might also add that Neil Hand is co-speaking with myself and Philip Seifert this year at the annual National Society for Histotechnology - Symposium/Convention in Vancouver B.C. Our workshop is titled: Resin Applications Forum: Methods for Processing, Special Staining, Immunohistochemical and In Situ Hybridization of Soft and Hard Tissue Including Medical Device Implants During the last 50 years, numerous histological procedures have been described on resin embedded tissue. While different types of resins are available for different purposes, the acrylics provide the widest range of techniques, especially for light microscopy applications. However, as demand from HE to more sophisticated techniques increases, so too have the problems, and nowhere is this more apparent and controversial than in the application of immunohistochemistry on resin sections. This workshop will provide a review and discussion for those individuals that currently work with and/or are just getting started working with soft and hard tissue specimens and specifically the various resins (i.e. MMA, GMA, Technovit, Acrylosin, etc.) associated with their specific tissue interests. The workshop will also detail the preparation and staining of sections of soft and hard tissue, including implants (e.g. undemineralized bone and cardiovascular stents), for immunohistochemical and in situ hybridization staining using different acrylic and epoxy resin embedding media. Specific problems and pitfalls, either technical or operational associated with certain resin embedding procedures, will be illustrated and examined. Particular emphasis will be given to procedures which have been used extensively for routine diagnostic, and research purposes, i.e. those that WORK! Individuals with a current or future intent to process and cut undemineralized tissue or tissue containing foreign implant materials using acrylic or epoxy resins are strongly encouraged to attend this workshop! Please feel free to contact me if you would like more information about the workshop as information relevant to the exact date and time becomes available. All I know at this time is that the NSH meeting is September 29th - October 3rd, 2012. Best Regards, Jack Jack Ratliff Hard Tissue Histologist Chairman, Hard Tissue Committee - National Society for Histotechnology From: gayle.cal...@bresnan.net To: histonet@lists.utsouthwestern.edu Date: Mon, 12 Mar 2012 11:04:20 -0600 Subject: [Histonet] Re: undecalcified bone IHC Jeff, It is most certainly possible to do IHC on undecalcifed bone sections embedded in PMMA although not the easiest task. Sectioning is done on a microtome that is powerful enough to cut the plastic and using tungsten carbide knives. The key is total removal of the plastic from MMA embedded bone sections to allow antibody/ immunoglobulins to access antigenic sites. Neil Hand has done IHC successfully on PMMA embedded tissues including undecalcified bone on 2 to 3 µm thick sections. I think one could cut thicker sections at 4 to 5 µm and still be successful. I do not recall what Troiano et al used. The following publications will help you and should include protocols, although conventional protocols will work according to Hand. Blythe D. Hand N et al 1997 J Clin Path 50:45-49. The use of methyl methacrylate resin for embedding bone marrow trephine biopsies. Hand NM et al 1996 Antigen unmasking using microwave heating on formalin fixed tissue embedded in methyl methacrylate J Cellular Path 1:31-37 Jackson P et al. 1996 Amplification of immunocytochemical reactions by the catalytic deposition of biotin on tissue sections. J Path 170(suppl):23A. This was about tyramide amplification when one gets a weak signal from conventional methods. Hand NM, Church RJ 1998 Superheating using pressure cooking: its use and application in unmasking antigens embedded in methyl methacrylate. J Histotechnology 2`:231-236 Hand NM et al 1989 Immunohistochemistry on resin embedded tissue for light microscopy: a novel post embedding procedure. Proceeding Royal Microscopical Society 24(1):A54-55. Hand NM Plastic Embedding media and techniques, Ch.30, p 663-677. Theory and Practice of Histological Technique, 5th edition by Gamble and Bancroft. The 6th edition is updated under same title. Use Google Scholar to find Troiano N et al from Yale on doing IHC on PMMA embedded bone sections with publications in J Histotechnology. Hand mentioned several HIER methods, using citrate buffer. Optimizing retrieval will depend on the antigen and you may end up doing this with some form of HIER, including microwave or other heat producing
Re: [Histonet] Re: undecalcified bone IHC
Thank you Gayle. Vikki On Mar 12, 2012 1:04 PM, gayle callis gayle.cal...@bresnan.net wrote: Jeff, It is most certainly possible to do IHC on undecalcifed bone sections embedded in PMMA although not the easiest task. Sectioning is done on a microtome that is powerful enough to cut the plastic and using tungsten carbide knives. The key is total removal of the plastic from MMA embedded bone sections to allow antibody/ immunoglobulins to access antigenic sites. Neil Hand has done IHC successfully on PMMA embedded tissues including undecalcified bone on 2 to 3 µm thick sections. I think one could cut thicker sections at 4 to 5 µm and still be successful. I do not recall what Troiano et al used. The following publications will help you and should include protocols, although conventional protocols will work according to Hand. Blythe D. Hand N et al 1997 J Clin Path 50:45-49.The use of methyl methacrylate resin for embedding bone marrow trephine biopsies. Hand NM et al 1996 Antigen unmasking using microwave heating on formalin fixed tissue embedded in methyl methacrylate J Cellular Path 1:31-37 Jackson P et al. 1996 Amplification of immunocytochemical reactions by the catalytic deposition of biotin on tissue sections. J Path 170(suppl):23A. This was about tyramide amplification when one gets a weak signal from conventional methods. Hand NM, Church RJ 1998 Superheating using pressure cooking: its use and application in unmasking antigens embedded in methyl methacrylate. J Histotechnology 2`:231-236 Hand NM et al 1989 Immunohistochemistry on resin embedded tissue for light microscopy: a novel post embedding procedure. Proceeding Royal Microscopical Society 24(1):A54-55. Hand NM Plastic Embedding media and techniques, Ch.30, p 663-677. Theory and Practice of Histological Technique, 5th edition by Gamble and Bancroft. The 6th edition is updated under same title. Use Google Scholar to find Troiano N et al from Yale on doing IHC on PMMA embedded bone sections with publications in J Histotechnology. Hand mentioned several HIER methods, using citrate buffer. Optimizing retrieval will depend on the antigen and you may end up doing this with some form of HIER, including microwave or other heat producing methods and with different buffers. Enzyme digestion is also a possibility. Hand removed MMA with xylene, warm my speed up the removal, also more than one change for 10 - 20 minutes or longer. When I talked to him personally, he said he had used warm xylene although temperature was not mentioned in his chapter. After MMA removal, rehydrate section through alcohol gradient as one does paraffin sections.He was emphatic about never allowing the sections dry out. Hopefully Jack Ratliff and Damien Laudier will provide more insight on this topic. Good luck Gayle M. Callis HTL/HT/MT(ASCP) ** Hi Jeff, If is it possible a few more specifics of how the tissue has been received, processed and evaluated would help. Undecalcified bone sectioning procedures vary and also what specific markers are you looking to do is important. Vikki On Mon, Mar 12, 2012 at 11:06 AM, Rene J Buesa rjbuesa @t yahoo.com wrote: Undecalcified? How are you going to section it? If you can section it, just use any IHC protocol for regular sections. Good luck! René J. --- On Mon, 3/12/12, Jeffery Howery Jeffery.Howery @t jcl.com wrote: From: Jeffery Howery Jeffery.Howery @t jcl.com Subject: [Histonet] Undecalcified bone IHC To: histonet @t lists.utsouthwestern.edu Date: Monday, March 12, 2012, 10:59 AM Does anyone have a protocol for Undecalcified bone for IHC? ___ 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
