Re: [Histonet] Processing Schedule- ASP-6025
Processing seems adequate. After processing, how long do they sit in the embedding centre block holding tank before embedding? We found that quite a few antigens were affected when we stored control tonsil in the embedding centre (dry) at 60oC for a few days before embedding. In summary: AntibodyClone Dried (Normal = 3+) CD4 4B120 BOB-1 TG140 CD3 LN101+ CD79a JCB117 1+ Oct-2 Oct-207 1+ CD8 4B112+ CD20L26 3+ So CD20 was unaffected but this process affected most of the antigens with some losing antigen recognition by the antibody (eg CD4 and BOB-1). Another one of those pre-analytical issues we need to consider. And yes I am writing this up for submission! Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) | Principal Scientist; Adjunct Fellow, School of Medicine, University of Western Sydney; Visiting Lecturer, School of Life Sciences, Faculty of Science, University of Technology Sydney | Histopathology t: (02) 9845 3306 | f: (02) 9845 3318 | e: tony.henw...@health.nsw.gov.au | w: www.schn.health.nsw.gov.au m: Cnr Hawkesbury Road and Hainsworth Street, Westmead, NSW Australia Locked Bag 4001, Westmead 2145, NSW Australia ♲ Please consider the environment before printing this email. -Original Message- From: Greg Dobbin via Histonet [mailto:histonet@lists.utsouthwestern.edu] Sent: Wednesday, 8 May 2019 5:07 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Processing Schedule- ASP-6025 Hello colleagues, I recently stained (IHC) a section of normal tonsil from another facility with p16 and the resulting stain was better than the same stain on a section of my labs own normal tonsil control. This has led us to question our processing schedule. I am not concerned with our fixation because we fix everything for at least 24 hours in 10% formalin (commercially prepared) prior to processing. Does anything jump out at you as being a potential red flag in the following overnight protocol? - Formalin 15 mins; RT - Processing water 1 min; RT - ETOH 70% 30 mins; 35C - ETOH 80% 30 mins; 35C - ETOH 95% 30 mins; 35C - ETOH 100% 30 mins; 35C - ETOH 100% 40 mins; 35C - ETOH 100% 60 mins; 35C - Xylene 60 mins; 35C - Xylene 60 mins; 35C - Xylene 60 mins; 35C - Paraffin 60 mins; 57C; vacuum - Paraffin 60 mins; 57C; vacuum - Paraffin 60 mins; 57C; vacuum Our formalin is changed after every 1100 cassettes and the alcohol, xylenes and paraffins are managed similarly by the instrument. Our specimen mix is a little of everything (skins, GIs, breasts, needle cores, gall bladders, gyne, etc). The one unknown (so far) in this story, is how the tonsil from the other laboratory was handled (ie the fixative used and for how long-I am assuming 10% formalin). Obviously, many of you will have schedules that differ from this one, in any number of ways, but what I am looking for from you is your opinion: *is there anything about this schedule that is particularly concerning?* Thank you, Greg -- *Greg Dobbin* 1205 Pleasant Grove Rd RR#2 York, PE C0A 1P0 *Everything in moderation...even moderation itself**!* ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This message is intended for the addressee named and may contain confidential information. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message are those of the individual sender, and are not necessarily the views of NSW Health or any of its entities. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Benchmarks for Reprocessing
Good afternoon Fellow histonetters! I was wondering if there are any tertiary institutions out there that have set a benchmark for reprocessing tissue? We tend to be less than 1%, but would love to see what others think is reasonable. Thanks for the feedback and hope you all are having an excellent day! Cristi ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Processing Schedule- ASP-6025
Hello colleagues, I recently stained (IHC) a section of normal tonsil from another facility with p16 and the resulting stain was better than the same stain on a section of my labs own normal tonsil control. This has led us to question our processing schedule. I am not concerned with our fixation because we fix everything for at least 24 hours in 10% formalin (commercially prepared) prior to processing. Does anything jump out at you as being a potential red flag in the following overnight protocol? - Formalin 15 mins; RT - Processing water 1 min; RT - ETOH 70% 30 mins; 35C - ETOH 80% 30 mins; 35C - ETOH 95% 30 mins; 35C - ETOH 100% 30 mins; 35C - ETOH 100% 40 mins; 35C - ETOH 100% 60 mins; 35C - Xylene 60 mins; 35C - Xylene 60 mins; 35C - Xylene 60 mins; 35C - Paraffin 60 mins; 57C; vacuum - Paraffin 60 mins; 57C; vacuum - Paraffin 60 mins; 57C; vacuum Our formalin is changed after every 1100 cassettes and the alcohol, xylenes and paraffins are managed similarly by the instrument. Our specimen mix is a little of everything (skins, GIs, breasts, needle cores, gall bladders, gyne, etc). The one unknown (so far) in this story, is how the tonsil from the other laboratory was handled (ie the fixative used and for how long-I am assuming 10% formalin). Obviously, many of you will have schedules that differ from this one, in any number of ways, but what I am looking for from you is your opinion: *is there anything about this schedule that is particularly concerning?* Thank you, Greg -- *Greg Dobbin* 1205 Pleasant Grove Rd RR#2 York, PE C0A 1P0 *Everything in moderation...even moderation itself**!* ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] time for penetration of methacarn fixative
John, You are such a wealth of information. I look forward to your posts. I learn something every time. That is going to be my reading list as well. Thank you. Betsy Molinari HT (ASCP) Texas Heart Institute 6770 Bertner Ave Houston, TX 77030 832-355-6524 (lab) 832-355-6812 (fax) Betsy Molinari Sr. Histology Research Technician CV Pathology Texas Heart Institute 6770 Bertner Avenue, MC 1-283 Houston, TX 77030 Office: 832-355-6524 | Fax: 832-355-6812 Email: bmolin...@texasheart.org texasheart.org | facebook | twitter -Original Message- From: John Kiernan via Histonet [mailto:histonet@lists.utsouthwestern.edu] Sent: Sunday, May 05, 2019 11:26 PM To: pwno...@gmail.com Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] time for penetration of methacarn fixative *** Important*** This email is not from Texas Heart Institute. Only click links or open attachments you know are safe. Peter Noyce, you did not say what tissue(s) you are planning to fix, or how big the specimens will be. Carnoy (1886) and methacarn (1970) were developed for animal tissues, cleverly balancing the actions of acetic acid and an alcohol in the presence of a hydrophobic solvent (chloroform) that diluted both these ingredients and also enhanced permeation by dissolving lipids. Carnoy's and other acid-alcohol fixatives are also used for plant specimens, which respond differently. If you are working with plant specimens you probably have a book by Steven E. Ruzin (1999) ISBN 0195089561. For the actions of alcohols and acetic acid in fixation of animal tissues, consult any text book of histotechnology or histochemistry published since about 1950. For selfish reasons, I recommend ISBN 9781907904325 (published in 2015) as an item to buy for your lab's bookshelf. Answers to your question are discussed in Chapter 5. With methacarn and other alcohol-acetic fixatives, no slow chemical reactions are involved (an important difference from formaldehyde-containing mixtures). Complete penetration accomplishes the fixation. The volume of fixative and procedure for subsequent processing into paraffin are very important. You need to read the paper by Puchtler et al (1970) and follow the instructions exactly. Probably you should also read Puchtler et al (1968) to use this type of non-aqueous fixative intelligently. PhD students are intelligent. I have added a couple of more recent papers that relate to methacarn. Read Puchtler or a textbook first. Not all modern investigators (especially molecular biologists) understand what the ingredients of fixative mixtures do to the different components of cells and extracellular materials. Current papers with micrographs full of ghastly artifacts abound, even in journals with very high citation indices. There are published mixtures with names like "modified methacarn" that may be OK for extracting RNA but do not have ingredients in correct proportions for minimizing distortion. Here's your list of recommended readings. - - - - - Puchtler H, Waldrop FS, Meloan SN, Terry MS, Connor HM (1970) Methacarn (methanol-Carnoy) fixation. Practical and theoretical considerations. Histochemie 21: 97-116. Puchtler H, Waldrop FS, Conner HM, Terry MS (1968) Carnoy fixation: practical and theoretical considerations. Histochemie 16: 361-371. Uneyama C, Shibutani M, Masutomi N, Takagi H, Hirose M (2002) Methacarn fixation for genomic DNA analysis in microdissected paraffin-embedded tissue specimens. J. Histochem. Cytochem. 50: 1237-1245. Buesa RJ (2008) Histology without formalin? Ann. Diagn. Path. 12: 387-396. - - - - - I wish you well with your research, and hope you will get your PhD while you are still young. John Kiernan London, Canada https://urldefense.proofpoint.com/v2/url?u=https-3A__www.schulich.uwo.ca_anatomy_people_bios_emeriti_kiernan-5Fjohn.html=DwICAg=5rShgjAa2OW9AX8kjI_BFTEsAS2aUiDYBgABvVqRiz0=4JF0M5k_UrXYJLzefN3bjagdyUrCioVawjbCC16NNH8=WmaHc4fCJS42J-KsPVJSnE9N1rCFdozPF9DSJA90hT8=v6m2a9-Ol7YLjPL_ZAaTvLmF4mPHwe1z57iL7Vo_VAI= https://urldefense.proofpoint.com/v2/url?u=http-3A__biostain.com=DwICAg=5rShgjAa2OW9AX8kjI_BFTEsAS2aUiDYBgABvVqRiz0=4JF0M5k_UrXYJLzefN3bjagdyUrCioVawjbCC16NNH8=WmaHc4fCJS42J-KsPVJSnE9N1rCFdozPF9DSJA90hT8=QPHVD0tPdeuvGeqJKyIsaA2Vv7HHNSCMi8LZw2MgKHE= = = = From: peter noyce via Histonet Sent: 03 May 2019 19:47 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] time for penetration of methacarn fixative Does any one have data for the time it takes for methacarn fixative (60% methanol, 30% chloroform, 10% glacial acetic acid) to penetrate and then fully fix tissue? PW Noyce -PhD student _ Histonet mailing list Histonet@lists.utsouthwestern.edu
