Re: [Histonet] B-gal positive control
Amos, hello. Do you have a reference for this? All my files talk about endogenous B-gal in kidney and pancreas and other organs (but then article talks about after lacZ transgenic manipulation) or demonstration of alpha-galactosidase in kidneys or in senescence associated or lysozomal storage diseases or differentiating light background staining from lacZ with pH tricks or pictures of islet cells staining with surrounding exocrine pancreas staining or just the journalistic form of hand-waving data not shown. Is beta-galactosidase readily expressed in completely normal kidney and where specifically? Thanks, Ray - Original Message - From: Amos Brooks amosbro...@gmail.com To: histonet@lists.utsouthwestern.edu Sent: Sunday, May 10, 2015 8:52:45 AM Subject: [Histonet] B-gal positive control Hi, Normal kidney should work fine for this. Amos On Fri, May 8, 2015 at 1:00 PM, histonet-requ...@lists.utsouthwestern.edu wrote: Message: 9 Date: Fri, 8 May 2015 14:50:42 + From: Coffey, Anna (NIH/NCI) [C] anna.cof...@nih.gov To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Subject: [Histonet] B-gal positive control Message-ID: 5c3e10119a1b824fbe92b08279f74a9101799...@msgb10.nih.gov Content-Type: text/plain; charset=us-ascii Hello Histonet, Has anyone out there come across a good FFPE positive control for B-gal? If so, please let me know! We would like to purchase a block or unstained slides if at all possible. Thanks! Anna Anna Coffey, MS, HTL(ASCP)CM Histotechnologist ___ 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] B-gal positive control
Anna, Don't know if you are talking human or non-human control or if makes a difference and I don't want to get into that whole discussion again. But if a mouse control is OK, one of the cleanest and nicest systems I used for B-gal was to get a transgenic mouse, easily obtainable with a Tie-2/lacZ promoter/reporter. B-gal expressed only on vascular endothelium so your assay can easily be tweaked for strength and cleanliness of signal. Have all the frozen/FFPE blocks you could ever need. Ray, Lake Forest Park, WA - Original Message - From: Anna Coffey (NIH/NCI) [C] anna.cof...@nih.gov To: histonet@lists.utsouthwestern.edu Sent: Friday, May 8, 2015 7:50:42 AM Subject: [Histonet] B-gal positive control Hello Histonet, Has anyone out there come across a good FFPE positive control for B-gal? If so, please let me know! We would like to purchase a block or unstained slides if at all possible. Thanks! Anna Anna Coffey, MS, HTL(ASCP)CM Histotechnologist Center for Advanced Preclinical Research Frederick National Laboratory for Cancer Research Leidos Biomedical Research, Inc. Bld 539, 224 Frederick, Maryland 21702 anna.cof...@nih.gov 301-846-1730 ___ 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] Can't log into Histonet to do anything
I got your message Gayle (through Histonet) although haven't heard much at all weekend otherwise. Use IE. Ray in Lake Forest Park, WA - Original Message - From: Gayle Callis gayle.cal...@bresnan.net To: Histonet histonet@lists.utsouthwestern.edu Sent: Monday, May 4, 2015 3:21:01 PM Subject: [Histonet] Can't log into Histonet to do anything Dear Histonettters, At the risk of being pesky, is Histonet having problems. I generally go to Histonet via Firefox/Google and haven't been able to get to the website for two days. I only hope someone out there can even get this message. I have tried finding Marvin Hanna's email address. Stymied and dead in the water. I would love to unsubscribe, but not sure anyone gets this message. Gayle Callis ___ 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
[Histonet] histology in higher education
The following has to do with histology and STEM (science, technology, engineering, math) so if not interested, please ignore. But I believe it can have real meaning to the profession of histology at the NSH, state society and local levels. I am elected to the Board of WSSEF (Washington State Science and Engineering Fair) where I am in educational outreach and also the assistant to the head judge. We recently had our Washington State Fair with 650 kids, grades 1-12 from all over the state. And while there was a lot of engineering and robots and computers there were a few projects having to do with medicine, biotechnology, immunology and pathology with some familiar histology or immunohistochemistry pictures included. At the end of the fair, we awarded almost 1.8 MILLION dollars of scholarships and awards to grades 7-12 students. Not only that, our top winners get an all-expenses paid trip to present at the ISEF (Intel International Fair) with 1,700 students competing from all 50 states and 70 countries. Wherever you are in the US, you have a state fair. I would advocate for some of you so interested at the national, state or local levels to promote histology, by getting involved as mentors for middle and high school students to science fairs; especially those that could lead to histopathology or other related projects that could lead into Intel affiliated fairs resulting in great benefit to the student and a spread of the word of histology into both the STEM world and general population. I've mentored for 15 years. It can be done. Molecular histopathology, personalized diagnostics and therapeutics, advances in immunohistochemistry, current controversies about breast biopsy diagnosis, or other disease with newer classifications, PCR and RTPCR in histology, modern-targeted therapeutics like in melanoma or colo-rectal carcinoma, FISH, digital image analysis software for you computer geeks and on and on; the list is nearly limitless. Especially if you are close to or can contact biotech companies or educational institutions to find co-mentors for grades 7-12 there are histology-related science project possibilities in terms of data collection and the scientific method and project presentation are nearly unlimited now. Be a mentor for or engage a grade 7-12 student, with the help of another mentor or organization, to think about (histology-related) projects for science fairs leading to a state fair and Intel ISEF. Can't think of any better way to promote histology so would hope those at NSH would take note of this. And since the ISEF fair receives projects and groups from 70 countries, I hope any outside the US would also think about the same thing. Ray Koelling HT, HTL, QIHC, STEM educational outreach advocate Lake Forest Park, WA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] histology in higher education
Hi, Thanks for info and happy to hear that. Didn't realize. May 10-15 is Worlds largest International Science and Engineering Fair in Pittsburgh this year and maybe will see others there and watch a high school histology project from somewhere bring home a $75,000 scholarship. Ray - Original Message - From: Joelle Weaver joellewea...@hotmail.com To: koelli...@comcast.net, histonet@lists.utsouthwestern.edu Sent: Wednesday, April 22, 2015 11:53:14 AM Subject: RE: [Histonet] histology in higher education Many people do this, and have donated hundreds of hours of their own time. But definately not enough. Good to encourage people to get involved. Joelle Weaver MAOM, HTL (ASCP) QIHC Date: Wed, 22 Apr 2015 14:07:18 + From: koelli...@comcast.net To: histonet@lists.utsouthwestern.edu Subject: [Histonet] histology in higher education The following has to do with histology and STEM (science, technology, engineering, math) so if not interested, please ignore. But I believe it can have real meaning to the profession of histology at the NSH, state society and local levels. I am elected to the Board of WSSEF (Washington State Science and Engineering Fair) where I am in educational outreach and also the assistant to the head judge. We recently had our Washington State Fair with 650 kids, grades 1-12 from all over the state. And while there was a lot of engineering and robots and computers there were a few projects having to do with medicine, biotechnology, immunology and pathology with some familiar histology or immunohistochemistry pictures included. At the end of the fair, we awarded almost 1.8 MILLION dollars of scholarships and awards to grades 7-12 students. Not only that, our top winners get an all-expenses paid trip to present at the ISEF (Intel International Fair) with 1,700 students competing from all 50 states and 70 countries. Wherever you are in the US, you have a state fair. I would advocate for some of you so interested at the national, state or local levels to promote histology, by getting involved as mentors for middle and high school students to science fairs; especially those that could lead to histopathology or other related projects that could lead into Intel affiliated fairs resulting in great benefit to the student and a spread of the word of histology into both the STEM world and general population. I've mentored for 15 years. It can be done. Molecular histopathology, personalized diagnostics and therapeutics, advances in immunohistochemistry, current controversies about breast biopsy diagnosis, or other disease with newer classifications, PCR and RTPCR in histology, modern-targeted therapeutics like in melanoma or colo-rectal carcinoma, FISH, digital image analysis software for you computer geeks and on and on; the list is nearly limitless. Especially if you are close to or can contact biotech companies or educational institutions to find co-mentors for grades 7-12 there are histology-related science project possibilities in terms of data collection and the scientific method and project presentation are nearly unlimited now. Be a mentor for or engage a grade 7-12 student, with the help of another mentor or organization, to think about (histology-related) projects for science fairs leading to a state fair and Intel ISEF. Can't think of any better way to promote histology so would hope those at NSH would take note of this. And since the ISEF fair receives projects and groups from 70 countries, I hope any outside the US would also think about the same thing. Ray Koelling HT, HTL, QIHC, STEM educational outreach advocate Lake Forest Park, WA ___ 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] controls to lengthy off topic
Hello Garrey, Curious myself, CAP contact info seems to be greyed out on website unless I officially log in and for now my concerns are with the Washington State Science and Engineering Fair for K-12 and golf game. (1) There are at least two phrases in the ANP.21450 which could be parsed out similar to the now famous it depends on what the definition of is is. (2) Fortunate, I had micro groups around who could provide me with species specific Candida or Aspergillus or species and morphological identifiable gram positive or gram negative organisms so when I built the controls with fresh human tissue, as has been described several times on Histonet by others, I knew exactly what I was looking at. (3) It appears there may be tens to hundreds of thousands of molds and what is growing in orange peels or strawberries or cream cheese or bacteria in slim jims would be a total mystery but maybe that is OK. Yet, human pathogen or not? rare or common? stains appropriately or not according to what it REALLY is? I'm not saying the controls are wrong; they might be perfectly fine. I'm just curious if anyone being inspected ever put a stained section of a slim jim on scope in front of a Pathologist from the inspecting agency and what was the reaction if any. Ray in Lake Forest Park, WA - Original Message - From: Garrey Faller garr...@gmail.com To: koelli...@comcast.net Cc: tjfinney2...@gmail.com, histonet@lists.utsouthwestern.edu Sent: Monday, April 20, 2015 3:50:15 PM Subject: Re: [Histonet] (no subject) Here is the CAP checklist requirement: ANP.21450 All histochemical stains are of adequate quality, and daily controls are demonstrated on each day of use for the tissue components or organism for which they were designed. Ray...you should call the CAP and ask for guidance on this. My interpretation of this requirement is that it should be OK to use a fungus from an orange peel. An orange peel fungus should have the same staining characteristics as a candida or aspergillus etc. Similarly a bacteria is a bacteria. If you can produce a control that has both gram positives and negatives, it should be OK. But, don't quote me on this. Call the CAP for a definitive answer. I am interested in their response. Garrey On Sun, Apr 19, 2015 at 9:06 PM, koelli...@comcast.net wrote: I asked about this in a different vein months ago. Has anyone shown a strawberry or ground meat or slim jim or orange peel as a bacteria/fungus control used for diagnostics to an inspector inspecting the lab and was there any comment from the inspector either positive or negative. Never heard back anything. Ray, Lake Forest Park, WA - Original Message - From: tjfinney2...@gmail.com To: histonet@lists.utsouthwestern.edu Sent: Sunday, April 19, 2015 5:24:53 PM Subject: [Histonet] (no subject) GMS controls From my understanding we can't use non human controls on patients. I could be wrong, but you may want to look into it. Happy Connecting. Sent from my Sprint Phone. ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] (no subject)
I asked about this in a different vein months ago. Has anyone shown a strawberry or ground meat or slim jim or orange peel as a bacteria/fungus control used for diagnostics to an inspector inspecting the lab and was there any comment from the inspector either positive or negative. Never heard back anything. Ray, Lake Forest Park, WA - Original Message - From: tjfinney2...@gmail.com To: histonet@lists.utsouthwestern.edu Sent: Sunday, April 19, 2015 5:24:53 PM Subject: [Histonet] (no subject) GMS controls From my understanding we can't use non human controls on patients. I could be wrong, but you may want to look into it. Happy Connecting. Sent from my Sprint Phone. ___ 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] Re: TRAP staining on formic acid decalcified bone reference
Hi all, as usual Gayle was right on. Use a buffered (more gentle) formic acid; not just formic acid per se of any water diluted concentration. For end point testing, critical, we used a radiograph machine instead of chemical endpoints which is also fine;we just had access to a lot of equipment. Ray Washington - Original Message - From: Gayle Callis gayle.cal...@bresnan.net To: Histonet histonet@lists.utsouthwestern.edu Sent: Thursday, April 2, 2015 3:12:55 PM Subject: [Histonet] Re: TRAP staining on formic acid decalcified bone reference The reference within a reference from Ray is A Chimeric Form of Osteoprotegerin Inhibits Hypercalcemia and Bone Resorption Induced by IL-1β, TNF-α, PTH, PTHrP, and 1,25(OH)2D3 . Sean Morony et al . J Bone Mineral Res V 14, pp 1478-1485. However, the formic acid decalcification method is not described in detail and merely says formic acid but whether this is buffered formic acid or just dilute formic acid in water only is not stated. Ray might elaborate on what specific formic acid recipe he used as many in research don't always use buffered formic acid decalcifiying solutions. I would assume Morony et all used a buffered formic acid with either sodium formate or sodium citrate and controlled so as to not overexpose TRAP to acids longer than necessary. One publication, i.e., Eggert and Germain. Stable Acid Phosphatase I. Demonstration and Distribution. Histochem 66, pp 301-317, 1980) discussed in detail the rapid demineralization in acidic buffers i.e. buffered formic acid for staining of stable forms of acid phosphatase. I have both of these publications on file and will forward privately. I would err on the side of using a buffered formic acid with either sodium formate or sodium citrate for doing this and use decalcification endpoint testing to avoid over exposure to acid i.e. over decalcification. Take care Gayle M. Callis HTL/HT/MT(ASCP) ___ 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] RE:Question about Formic acid decal and TRAP stain
Debra, it appears most of the histology world disagrees with me but I stand by my post. If TRAP didn't work with formic acid in our hands, a major pharmaceutical treatment wouldn't be ready to help women with post-menopausal osteoporosis. Ray - Original Message - From: Sarah Mack sarah_m...@urmc.rochester.edu To: histonet@lists.utsouthwestern.edu Sent: Thursday, April 2, 2015 10:21:21 AM Subject: [Histonet] RE:Question about Formic acid decal and TRAP stain In our hands we have never had TRAP success on Formic acid decalcified tissue. Sarah Mack University of Rochester Medical Center Center for Musculoskeletal Research Histology, Biochemistry, and Molecular Imaging Core 601 Elmwood Avenue Box 665 Rochester, NY 14642 (585)-273-3901 From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of histonet-requ...@lists.utsouthwestern.edu [histonet-requ...@lists.utsouthwestern.edu] Sent: Thursday, April 02, 2015 1:02 PM To: histonet@lists.utsouthwestern.edu Subject: Histonet Digest, Vol 137, Issue 3 Send Histonet mailing list submissions to histonet@lists.utsouthwestern.edu To subscribe or unsubscribe via the World Wide Web, visit https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.utsouthwestern.edu_mailman_listinfo_histonetd=AwIDaQc=4sF48jRmVAe_CH-k9mXYXEGfSnM3bY53YSKuLUQRxhAr=ODw50OyFtFWu8REOenc_8wsdRMG_cbkreuWUix7iMVom=KnPSEWHFntXYp-Xo0ibYtyMEBbK1UL1yi6Dm03IvZLEs=OUpH8OsAE5aX_ZTdjsfvOwqMMBDeESpHHBEfWaX3Fzge= or, via email, send a message with subject or body 'help' to histonet-requ...@lists.utsouthwestern.edu You can reach the person managing the list at histonet-ow...@lists.utsouthwestern.edu When replying, please edit your Subject line so it is more specific than Re: Contents of Histonet digest... Today's Topics: 1. RE: Allowable temperature range (Linda Prasad (SCHN)) 2. Positive Control for IF? (Paula Sicurello) 3. C3d? (Paula Sicurello) 4. Re: Positive Control for IF? (Mark Tarango) 5. Re: Positive Control for IF? (Jim Burchette) 6. RE: C3d? (Sebree Linda A) 7. interface between Bond and Cerner CoPath Plus (Nancy Schmitt) 8. Question about Formic acid decal and TRAP stain (Debra Siena) 9. RE: Question about Formic acid decal and TRAP stain (Elizabeth Chlipala) -- Message: 1 Date: Wed, 1 Apr 2015 22:39:00 + From: Linda Prasad (SCHN) linda.pra...@health.nsw.gov.au Subject: RE: [Histonet] Allowable temperature range To: 'Tim H' thiggin...@msn.com, histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: 1217ddb3d7de5e418e3d560a268eabd0e0e88...@xmdb03.nch.kids Content-Type: text/plain; charset=utf-8 If it's in formalin it can just stay at room temperature. Linda Prasad | Senior Scientist | Histopathology t: (02) 9845 3306 | f: (02) 9845 3318 | e: linda.pra...@health.nsw.gov.au | w: https://urldefense.proofpoint.com/v2/url?u=http-3A__www.schn.health.nsw.gov.aud=AwIDaQc=4sF48jRmVAe_CH-k9mXYXEGfSnM3bY53YSKuLUQRxhAr=ODw50OyFtFWu8REOenc_8wsdRMG_cbkreuWUix7iMVom=KnPSEWHFntXYp-Xo0ibYtyMEBbK1UL1yi6Dm03IvZLEs=9Qb6_bEDCJKheqNhwxTb8wBpygzmxDAIDXa9byoCD2ke= 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: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tim H Sent: Thursday, 2 April 2015 1:52 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Allowable temperature range What is the allowable temperature range for a histology specimen after collection in formalin for shipping and storage? Any ideas? Tim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.utsouthwestern.edu_mailman_listinfo_histonetd=AwIDaQc=4sF48jRmVAe_CH-k9mXYXEGfSnM3bY53YSKuLUQRxhAr=ODw50OyFtFWu8REOenc_8wsdRMG_cbkreuWUix7iMVom=KnPSEWHFntXYp-Xo0ibYtyMEBbK1UL1yi6Dm03IvZLEs=OUpH8OsAE5aX_ZTdjsfvOwqMMBDeESpHHBEfWaX3Fzge= * 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
Re: [Histonet] Question about Formic acid decal and TRAP stain
Hi Debra, My experience differs from Elizabeth and others. Indeed have made thousands of mouse bone preparations with formic acid decaled sections. Here is an article that didn't copy paste so well: RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism Ji Li*, Ildiko Sarosi † , Xiao-Qiang Yan † , Sean Morony † , Casey Capparelli † , Hong-Lin Tan † , Susan McCabe*, Robin Elliott*, Sheila Scully Gwyneth Van † , Stephen Kaufman † , Shao-Chieh Juan † , Yu Sun † , John Tarpley † , Laura Martin † , Kathleen Christensen James McCabe † , Paul Kostenuik † , Hailing Hsu*, Frederick Fletcher † , Colin R. Dunstan † , David L. Lacey , and William J. Boyle* Departments of *Cell Biology and Pathology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320 Edited by David V. Goeddel, Tularik, Inc., South San Francisco, CA, and approved December 20, 1999 (received for review September 30, 1999) PNAS u February 15, 2000 u vol. 97 u no. 4 u 1571 Is in PNAS and while the methods are not here in this article, look in the reference section to materials and methods used and see beautiful pictures of TRAP from formic acid decaled bones. I did them for years after that. Complete fixation and gentle formic acid (or immunocal) and indeed this can be done. Ray (retired in Lake Forest Park, WA, golf and science education outreach for K-12 busy) - Original Message - From: Debra Siena dsi...@statlab.com To: histonet@lists.utsouthwestern.edu Sent: Thursday, April 2, 2015 9:00:25 AM Subject: [Histonet] Question about Formic acid decal and TRAP stain Hi Histonetters, I have a question to ask if you don't mind. Can TRAP Histochemical staining be performed after decalcifying with formic acid? Any tricks of the trade, etc? If anyone has any experience or references that they could point me to, I would greatly appreciate it. Thanks in advance for your help. Debbie Siena dsi...@statlab.com mailto:bbro...@statlab.com%7C | www.statlab.comhttp://www.statlab.com/ ___ 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] RE: Mushrooms for GMS fungus control
Apparently there are numerous interesting ways for fungus or bacteria controls to be had from orange peels to hamburger to slim Jim's to hot dogs to strawberries to . Sounds like fun to me. I'm curious, with the emphasis now on quality control in labs run amok, has anyone passed a rigorous inspection actually showing these as your currently in-use controls? A PI in research who doesn't want his paper rejected at peer review. A CAP inspector in clinical labs who is nit-picky reviewing staining controls but might be looking for a phase anything deficiency. The dot-your-i's and cross-your-t's FDA people who might or might not OK your drug in development. Really, just curious if anyone with a hammer over your head has said it is perfectly fine to use them. Ray, Seattle, WA - Original Message - From: Linda Prasad (SCHN) linda.pra...@health.nsw.gov.au To: Jeffrey Robinson jrobin...@pathology-associates.com, histonet@lists.utsouthwestern.edu Sent: Sunday, March 8, 2015 4:09:02 PM Subject: [Histonet] RE: Mushrooms for GMS fungus control I used strawberries for a fungal control. Worked really good. Linda Prasad | Senior Scientist | Histopathology t: (02) 9845 3306 | f: (02) 9845 3318 | e: linda.pra...@health.nsw.gov.au | w: www.schn.health.nsw.gov.au 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: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jeffrey Robinson Sent: Saturday, 7 March 2015 4:16 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Mushrooms for GMS fungus control How about mushrooms? Has anyone had any success using mushrooms as a GMS fungus control? Jeff Robinson, Senior Histotechnologist, Sierra Pathology Lab, Clovis, CA This email and attachments may contain PHI that is privileged and confidential and is not intended for any unauthorized person. If you, the reader, are not the intended recipient you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. Do not read the email but instead reply to the sender and destroy the message and any attachments. Thank you. ___ 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] RE: Know Error
I am not disagreeing nor am I sticking up for the company and not sure I'd even agree with the company but I think there is much more to this, from what little I know of them, than just mixing up two specimens at a physicians office. I believe their point of view of the company is besides patient ID errors that in making a cancer diagnosis, that the cancer material came EXCLUSIVELY from that one patient and is not a contaminant from another real cancer patient. Think floaters upon cutting, think leftover friable material from an embedding well that ends up in a cassette because embedder didn't clean forceps. You PCR up a V600E BRAF mutation from a melanoma slide and tube and how do you know 100% it is not DNA from a different patient who really needs the therapeutic but the first patient doesn't. Is the mutation really representative of the first patient or just a contaminant from the second? Again, I truly wonder about this being useful but you have to admit, there is more than only patient mis-identification but there are floaters and contaminations that must be paid attention to. Just my opinion. Ray Seattle, WA - Original Message - From: Thomas S. Webster twebs...@crh.org To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Tuesday, February 3, 2015 11:52:13 AM Subject: [Histonet] RE: Know Error Most problems are with the physician offices, not the lab, because they have bad practices like pre-labeling specimens. How does this fix that problem? You could have a buccal specimen and biopsy that match up fine but are on the wrong patient. Seems like collecting a buccal smear just adds even more variables and opportunities for error. Spend that time properly labeling the ACTUAL specimen(s) and problem solved. There is at least one lab that is offering Know Error testing on abnormal pap tests believe it or not. I didn't realize an abnormal pap test lead immediately to a hysterectomy.. http://manhattanlabs.com/for-doctors/mypap/ CONFIDENTIALITY NOTICE: This e-mail message, including all attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. You may NOT use, disclose, copy or disseminate this information. If you are not the intended recipient, please contact the sender by reply e-mail immediately. Please destroy all copies of the original message and all attachments. Your cooperation is greatly appreciated. Columbus Regional Hospital 2400 East 17th Street Columbus, Indiana 47201___ 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] Achieving Better Antibody Penetrance in my Tissue Sections (IHC)
Michael, Not sure from your explanation if your mCherry tag is less intense on the inside of sections or if you are referring to the P and RFP Alexa and Cy5 stain intensity If the former, not sure about that. If the later, having done very similar thing in grad school, but with different tissue and molecule targets and different antibodies, your protocol looks very similar to what I did for years. With exception that I extended those secondaries time-wise. Whether right or wrong, my thinking that if it took 'overnight/shaken for a 150kDa antibody to penetrate through 50u (which is what I used), it would take more than just 2 hours for an even bigger molecule (Ab+tag) to penetrate. I'd try extending secondary times while keeping everything else same and look for better penetration. Otherwise, our protocols would look nearly identical. Ray Koelling Lake Forest Park, WA - Original Message - From: Michael Bruno mbru...@buffalo.edu To: histonet@lists.utsouthwestern.edu Histonet Listserve histonet@lists.utsouthwestern.edu Sent: Monday, January 19, 2015 11:53:01 AM Subject: [Histonet] Achieving Better Antibody Penetrance in my Tissue Sections (IHC) Hi All, I'm new to graduate school and the world of immunohistochemistry. I am looking for a way to change my protocols so that the tissue sections are stained/penetrated by the antibodies more effectively. I am doing an anti-substance P stain, followed by an anti-RFP stain in 50um sections of rat brains. The rats were injected unilaterally with an AAV10 (with an mCherry tag) virus (into the dorsal striatum) that was designed by my professor. When I observed the sections on a confocal microscope, it was apparent that the center of the sections do not have the same intensity of staining that is found on the outside edges of the tissue. == * Here is an outline of my procedure : * Take tissue sections I am interested in (previously observed for native fluorescence) * Rinse the floating sections in ~10mL PBS 3x5 minutes * Rinse the floating sections in ~10mL 0.5% PBST (Triton X) 3x5 minutes * Block sections in 5% normal goat serum (NGS), with 0.25% PBST for 1 hour @ room temp * Incubate sections in anti-substance P (anti-SP) at a [1:400] in 2.5% NGS, in 0.25% PBST. Overnight at 4*C, shaken. * o I used a 24-well plate to hold the sections separately. Each well contained 300ul of the solution described. I had 10 sections, and some controls. So 8 sections at a time would be treated with the antibodies. * Sections rinsed in PBS 3x5 minutes * Sections incubated in secondary antibody (Ab), Alexa488 [1:1000] in 0.25% PBST for 2 hours at room temp. * The the tissue sections are rinsed in PBS 4x10 minutes to remove extra antibodies. * At this point I start the second primary incubation: anti-RFP biotin conjugated [1:2400] in 0.25% PBST, overnight in the cold room. * Rinse sections in PBS 3x5 minutes * Incubate the sections in the second secondary antibody, Cy5 [1:2000] in 0.25% PBST for 2 hours at room temp. * Rinse the sections in PBS, 3x5 minutes. * Then the sections were mounted to glass slides. Prolong Gold w/ DAPI was the mounting medium. Cover slipped. * I dried for a few days, in a desk drawer; covered and dark. == Thanks for reading all of this. Since it would be impossible to change the thickness of the sections (they are cut and stored in the fridge in 0.02% sodium azide), I think I need to change incubation times, temperature, and PBST concentrations. Any tips would be greatly appreciated! If you have had a similar issue in the past, I would like to know what you did to fix it. Thanks! Mike ___ 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] X-gal staining
Kristopher, beta-gal is an enzyme and as far as I know is rendered inert after FFPE. I did do fresh skin in X-gal to target, as you would with any whole mount specimen, THEN FFPE and cut sections to it to see the signal. If that is not an option, go after it with an anti-beta galactosidase antibody (polyclonal-like Pierce I used-have zero relationship to them) from several places. Make sure the immunogen is a full, length protein that produced the antibody, and it will go much easier. Ray Raymond Koelling Seattle, WA area - Original Message - From: Kristopher Kalleberg kristopher.kalleb...@unilever.com To: histonet-requ...@lists.utsouthwestern.edu, histonet@lists.utsouthwestern.edu Sent: Thursday, January 8, 2015 9:53:38 AM Subject: [Histonet] X-gal staining Hello All, Does anyone know of a commercially available X-gal stain that would work on formalin fixed paraffin embedded skin samples in order to detect beta galactosidase. Thank you in advance. Kris ___ 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] PASD muscle stains
Tiffany, Have used 10%NBF on muscles but also alcoholic fixatives -alcoholic formalin or absolute- just always preferred 10%NBF since it gave the morphology and counterstaining I wanted. diastase in a 6.0pH buffer (don't heat above 40 degrees if trying to speed up heating-kill the diastase) and always stayed away from di water on frozen sections. di water too variable and fickle. Ray Koelling Lake Forest Park - Original Message - From: Tiffany Passaro tpass...@cellnetix.com To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, January 5, 2015 3:22:32 PM Subject: [Histonet] PASD muscle stains Greetings, I am looking for fixatives that others are using in their labs for the PASD stain on fresh frozen muscle tissue. Currently we are fixing in 10% NBF. Thanks in advance for any info on this. Tiffany DISCLAIMER: This message is intended for the sole use of the addressee, and may contain information that is privileged, confidential and exempt from disclosure under applicable law. If you are not the addressee you are hereby notified that you may not use, copy, disclose, or distribute to anyone the message or any information contained in the message. If you have received this message in error, please immediately advise the sender by reply email and delete this message. ___ 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] Double Labeling EM Blocks
Neelam, If you are referring to immunolabelling for transmission electron microscopy, it is relatively simple to apply and this worked wonderfully for projects we did, to use 2 antibodies to two different epitopes but each antibody with a different size gold particle attached. The gold particles are between a few nanometers up to 30 or 40 nanometers in diameter which are attached to antibodies and so are easy to tell apart from one another. Ray Lake Forest Park, WA - Original Message - From: Neelam Joshi neelam.jo...@nephropath.com To: histonet@lists.utsouthwestern.edu Sent: Thursday, October 23, 2014 5:42:21 AM Subject: [Histonet] Double Labeling EM Blocks Hello, Does anyone has a procedure for double labeling EM Blocks with two identifier? Please let me know as soon as possible. Thanks you all! Neelam Joshi,HT ___ 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] RE: Double Labeling EM Blocks
Oh, oh, sorry. I goofed. I thought this double labeling was technical and not regulatory. Ray Lake Forest Park - Original Message - From: Timothy Morken timothy.mor...@ucsfmedctr.org To: Neelam Joshi neelam.jo...@nephropath.com, histonet@lists.utsouthwestern.edu Sent: Thursday, October 23, 2014 7:23:58 AM Subject: [Histonet] RE: Double Labeling EM Blocks Neelam, we don't have two ids on the EM blocks themselves, but we do print a label from our lab computer system that has patient name and case number that is applied to the box that holds the blocks. Tim Morken Supervisor, Histology, Electron Microscopy and Neuromuscular Special Studies UC San Francisco Medical Center San Francisco, CA CONFIDENTIALITY NOTICE: This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential, proprietary, and/or privileged information protected by law. If you are not the intended recipient, you may not use, copy, or distribute this email message or its attachments. If you believe you have received this email message in error, please contact the sender by reply email and destroy all copies of the original message. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Neelam Joshi Sent: Thursday, October 23, 2014 5:42 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Double Labeling EM Blocks Hello, Does anyone has a procedure for double labeling EM Blocks with two identifier? Please let me know as soon as possible. Thanks you all! Neelam Joshi,HT ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] RE: Recycled or not? NO PHI
Hi Joyce, Absolutely agree with recycling concept, value, money saved and no fumes in lab (if using newer models) and if used properly. I've always been curious about the concept of a lab recycler making xylene purer by distilling out isomers. Which unit do you have? meta-xylene is in great demand as a feedstock for plastic production. Since xylene(s) are a mixture of ortho-, meta- and para all of which differ in boiling points by just very few degrees, they are (near) impossible to separate out from one another by ordinary distillation and need multi-fractional set-ups with crystallization and absorption and catalytic beds. Manufacturers spend vast sums to do this and are always looking for a better way. What unit do you have? Have you had chromatography done on your (new) input and then output xylene. I've done it extensively for alcohol but never xylene. Thanks, Ray Seattle, WA - Original Message - From: Joyce K. Weems joyce.we...@emoryhealthcare.org To: histonet@lists.utsouthwestern.edu Sent: Thursday, June 26, 2014 8:33:04 AM Subject: [Histonet] RE: Recycled or not? NO PHI I have used recycled xylene since the mid-80s and the only problem is that it is purer than new xylene and can make biopsies crispy. (The isomers get distilled out.) We use new xylene on the biopsy processor. The recycler is in our lab and there are no fumes at all. Surely does save money. Joyce Weems Pathology Manager 678-843-7376 Phone 678-843-7831 Fax joyce.we...@emoryhealthcare.org www.saintjosephsatlanta.org 5665 Peachtree Dunwoody Road Atlanta, GA 30342 This e-mail, including any attachments is the property of Saint Joseph's Hospital and is intended for the sole use of the intended recipient(s). It may contain information that is privileged and confidential. Any unauthorized review, use, disclosure, or distribution is prohibited. If you are not the intended recipient, please delete this message, and reply to the sender regarding the error in a separate email. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of McAnn, Sherrian Sent: Thursday, June 26, 2014 11:20 AM To: Blazek, Linda; Podawiltz, Thomas; histonet@lists.utsouthwestern.edu Subject: RE: [EXTERNAL] [Histonet] RE: Recycled or not? NO PHI We routinely recycle both our alcohols and xylenes. They are checked for purity and with the alcohol the extra step of ensuring that we are getting the correct percentage (95%) recovered. We have never had any issues in any of our processors or stainers since using recycled reagents. We also have not had an issue with fumes. The recyclers nowadays are much better than their older versions and I think that sometimes prejudices come into play with the older techs like me who were around for the older models. P. S. We used to have to do ours on a hotplate with a large round glass ball and would have to clean the ball out. Those were not the good ole days. :) -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Blazek, Linda Sent: Thursday, June 26, 2014 9:43 AM To: Podawiltz, Thomas; histonet@lists.utsouthwestern.edu Subject: [EXTERNAL] [Histonet] RE: Recycled or not? NO PHI I agree with Tom. With the exception of self-inflicted issues we also have not had any issues with recycling our reagents. We check each batch as it is recycled. We also don't have a problem with fumes. (And our pathologists are fussy) -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Podawiltz, Thomas Sent: Thursday, June 26, 2014 10:34 AM To: Barbara Tibbs; Sanders, Jeanine (CDC/OID/NCEZID); histonet@lists.utsouthwestern.edu Subject: [Histonet] RE: Recycled or not? NO PHI We have never had an issue with either our recycled xylene or alcohol that was not self inflicted. When our system is running there are no fumes. Tom Podawiltz HT (ASCP) Histology Section Head LRGHealthcare Laconia, NH 03246 603-524-3211 ext: 3220 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Barbara Tibbs Sent: Thursday, June 26, 2014 9:06 AM To: Sanders, Jeanine (CDC/OID/NCEZID); histonet@lists.utsouthwestern.edu Subject: [Histonet] RE: Recycled or not? While I can agree that recycling alcohol and xylene is both environmentally and economically advantageous, technically it's awful. There's no way to make used alcohol and xylene as pure as it was originally. There's also the issue of fumes from recycling a solvent. The company I had used years ago swore that there were no fumes when using their machine but the personnel working in the laboratory would vigorously disagree. Barbara S. Tibbs Histology
Re: [Histonet] RE: Formalin in the OR
Heartbreakingly sad, I do not know where the current regulations are but safety, as Terri rightly pointed out, is an accident that did happen. Not an anecdote, you can look up March 1985, Jackson Memorial Hospital in Miami (years after I left). Patient went to surgery, had some cerebrospinal fluid (CSF) removed during operation but an UNMARKED container of gluteraldehyde (aldehyde) fixative got marked as CSF with all the comings and goings over many hours. When the CSF was set to be reinjected as replacement, the fixative got reinjected as replacement instead of his CSF. Patient obviously died. Can't believe that is the only actual safety issue that has ever cropped up with surgery and formalin. So maybe a warning for both; no unlabeled bottles and no fixative right in the actual surgery suite. Ray Seattle WA - Original Message - From: Terri Braud tbr...@holyredeemer.com To: histonet@lists.utsouthwestern.edu Sent: Friday, June 13, 2014 10:52:43 AM Subject: [Histonet] RE: Formalin in the OR Wow, this is such a safety issue with an accident waiting to happen. I totally agree with Peggy that Formalin should not be allowed in an OR room. Even a gallon spill would be cause to evacuate and can you imagine the consequences of that? We have a small room off of the OR suites stocked with a 5 gallon carboy over a 5 gal spill container Terri L. Braud, HT(ASCP) Anatomic Pathology Supervisor Holy Redeemer Hospital Laboratory 1648 Huntingdon Pike Meadowbrook, PA 19046 Ph: 215-938-3676 Fax: 215-938-3874 2. Re: Formalin in operating (surgery) rooms (Lee Peggy Wenk) -Original Message- From: Lee Peggy Wenk Sent: Friday, June 13, 2014 7:44 AM To: Candace J. Wagner ; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Formalin in operating (surgery) rooms I think this is mostly a safety issue, and suggest NOT allowing any amount of formalin in OR/surgery rooms. - CONFIDENTIALITY NOTICE: This E-Mail is intended only for the use of the individual or entity to which it was sent. It may contain information that is privileged and/or confidential, and the use or disclosure of such information may also be restricted under applicable federal and state law. If you received this communication in error, please do not distribute any part of it or retain any copies, and delete the original E-Mail. Please notify the sender of any error by E-Mail. Thank you for your cooperation. ___ 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
[Histonet] leaving histology question research is still an option
Alpha Histotech, wanted to be sure that I did NOT tell you to drop everything in life to look to research exclusively. So I cut and pasted this from my original message Research histology should not be overlooked I stand by that statement. I agree with Emily that funding in research is (stupidly for this nation) difficult. But it is not zero. PhD's do NOT saturate histotech jobs in research labs. There might be a few, maybe some, maybe a lot in some places but not all. No one, even a clinical lab, will guarantee that job for years and years. I know many histotech/non-histotech techs who have been through 5-6 different more molecular labs in the same building for over 30 years. So have 30 years seniority in the system. Grant runs out and you move to a different lab (and is way easier having had made connections in first lab). I made 3x in research industry then I could ever have made in clinical histo lab. Is not for everyone but also not something to dismiss as hopeless. If you ever do research, you will find that N of 1 is not reliable to base everything and every decision on. Best of luck. Search for those options several others have given but don't dismiss my suggestion outright. Ray in Seattle (histotech from 1960's who could only retire now because of Research histology) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Should I leave histology world
Alpha Histotech- I'll put in my few words even though I'm not active anymore and possibly from different perspective. But also using a few assumptions and if my assumptions are wrong then the rest of what I say is probably meaningless. Not IDíng your e-mail address but if you've worked 3 jobs nightshift including a large reference lab, do you live near a big city? And if so is it a city close to a college or university. Research histology should not be overlooked. You will find many molecular or other such non-histo labs that actually do some or even a lot of histology by non-histology personnel or lab workers. Sometimes it is OK, sometimes even great. Sometimes, and I witnessed it, it is at an embarrassing histo level. I can walk up or down university hallways and see a genetics lab or some other molecular lab and see a microtome or cryostat in there. Sometimes those PI's will send histo work to a core lab. Sometimes they don't want to pay per block so do it (and staining and IHC and FISH) themselves. Someone with even minimal wide-ranging histo experience might be welcomed. No timed block cutting counts. Learn some immunology, genetics, molecular techniques, comparative medicine, physiology, etc, etc along the way. Many places even pay for college level courses while employed there. Just a thought if you are near that kind of area. Ray in Seattle - Original Message - From: joelle weaver joellewea...@hotmail.com To: Timothy Morken timothy.mor...@ucsfmedctr.org, Alpha Histotech optimusprimehistot...@hotmail.com, histonet@lists.utsouthwestern.edu Sent: Tuesday, June 3, 2014 5:55:09 PM Subject: RE: [Histonet] Should I leave histology world It would be a shame to get discouraged now after all the time you have already put into histology. If you still want to work in histology, I might suggest you try to have a conversation with a manager, supervisor or lead tech and see if they are willing to support you. Tell them you want to spend more time cuting to be able to section with high quality at the rate that works for their productivity standards. If you present it as a win-win proposition, see what resources, people and time they are willing to chip in to help get where they would like you to be. Make some metric or rate to achieve in microtomy your goal for the year, and put it into writing ( good for all goals:). Or if that is too uncomfortable , approach someone individually whose microtomy skills you admire , and see if they are willing to provide some tips and guidance off work time. I also went through a NAACLS program. Still at my first real histology job , the realization that this was the actual training became apparent very quickly. I had moments of exhaustion and feeling overwhelmed, but I now feel I was also fortunate to work initially at a pretty high volume place. It was a great breaking in for embedding and microtomy. Luckily there were also some experienced techs there who saw how much I wanted to learn, and were willing to help me get better. The constructive criticism stung sometimes, but they did me a huge service. But believe me, not everyone was helpful or supportive along the way. Try to ignore those kind of people as much as possible. And I still get criticized sometimes, make mistakes, and I still have more to learn. But here are a couple of other options for you to consider before you decide to leave, and what I did to get more experience when in your situation more quickly; Take on a second histology job that targets specific skills, tissue, or techniques you want more experience in. Believe me I have been criticized and misunderstood for this choice s well many times, but personally I do not regret any of those experiences now. I also feel that small labs are nice to build well rounded skills since you are usually more of a jack of all trades and have less tendency to do one task over your whole shift from day to day. Sometimes you just have to identify the environment that is the right fit for you. Best of luck to you- and let us know how things turn out! Joelle Weaver MAOM, HTL (ASCP) QIHC From: timothy.mor...@ucsfmedctr.org To: optimusprimehistot...@hotmail.com; histonet@lists.utsouthwestern.edu Date: Tue, 3 Jun 2014 22:51:31 + Subject: RE: [Histonet] Should I leave histology world CC: Alpha, it is clear to me, after 30+ years in the field, that some are born with the ability to cut fast AND do well at it. The rest of us just have to work harder at developing that skill. But it does take bench time to do it. A recent cache is that it takes 10,000 hours to become an absolute expert at something - that's about 5 years full time work. And that's just one skill. It sounds like you need some good teachers (ie, those who like to teach and like having their students do well). That would be the highest priority if you want to stay in
Re: [Histonet] HA-tag antibody
Eva, Not sure you are going to find that much published on HA-tag antibodies. That 9 amino acid sequence used by most to tag; people are looking to identify the fusion partner of the tag and worry little about publishing on the tag itself. Same with the famous 8 amino acid sequence FLAG-tag. I agree with Anatoli Gleiberman with what he recommended. Also look at Cell Signalling; they have non-mouse antibodies to HA-tag for paraffin and have some pictures. But few people are concerned with publishing on a tag itself but with the partner in the fusion that they are after. Used HA and FLAG tags in paraffin often but that wasn't any publishable finding. Beware if your mouse has influenza in being sure what you are seeing. Ray Koelling Seattle, WA - Original Message - From: Eva Permaul e...@georgetown.edu To: histonet histonet@lists.utsouthwestern.edu Sent: Wednesday, May 21, 2014 6:53:51 AM Subject: [Histonet] HA-tag antibody Hello, I am looking for an HA-tag antibody to detect a protein labeled with HA in formalin fixed paraffin embedded tissues. Could anyone suggest a published antibody? The tissue of interest is mouse so I would prefer an antibody made in another species. I have found several antibodies but can't find them published. Thank you, Eva Georgetown University ___ 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
a few words-Re: [Histonet] Was told I am having bounce-backs-off topic
I got a bounce a week ago and contacted Linda M.in private and was deemed fairly innocuous but as with all such things even remotely suspicious I deleted it and never, ever look into such things. As it turns out, was talking a few days ago to a judge for my district STEM science fair who happens to be a computer security geek for an international company. We were just discussing security failures in the Internet opening everyone, EVERYONE, up to fraud and disaster. The Heart Bleed problem, just now Microsoft warned of its newly discovered hole in IE, the way hackers can now target companies through vending machines at work that are hooked to company computers to monitor usage, the great security worry about the electrical infrastructure, targeting of dumb (I guess people who have them say smart) phones. When I went to college and learned biology, food and water seemed to drive life and things like the Krebs citric acid cycle. Now apparently digital gadgets are a part of actual life. Not as a luxury or convenience but as part of life itself without which there is apparently death. As you sew, so shall you reap. I hope we all can remember that evolution of our analog human brains with all sorts of cell signaling molecules has taken millions of years. Can't be re-evolved in just a few years; if ever. Sent from my old, but hopefully safe, desktop PC, guarded by several layers of security walls. Ray in Seattle - Original Message - From: Pam Marcum mucra...@comcast.net To: Paula Pierce cont...@excaliburpathology.com Cc: Histonet Histonet@lists.utsouthwestern.edu, Pamela A Marcum pamar...@uams.edu Sent: Monday, April 28, 2014 1:21:46 PM Subject: Re: [Histonet] Was told I am having bounce-backs We don't use Yahoo and the servers are not linked to a service outside the Universi ty as message system. - Original Message - From: Paula Pierce cont...@excaliburpathology.com To: Pamela A Marcum pamar...@uams.edu, Akemi Allison akemiat3...@yahoo.com, Histonet Histonet@lists.utsouthwestern.edu Sent: Monday, April 28, 2014 3:15:05 PM Subject: Re: [Histonet] Was told I am having bounce-backs I have received the same message. The problem may be that my website is hosted by yahoo servers, even though yahoo is not in the name. Many facilities are blocking anything from yahoo, gmail, etc. because of potential viruses. Paula K. Pierce, HTL(ASCP)HT President Excalibur Pathology, Inc. 5830 N Blue Lake Dr. Please note new address! Norman, OK 73069 405-759-3953 Lab 405-759-7513 Fax www.excaliburpathology.com From: Marcum, Pamela A pamar...@uams.edu To: 'Akemi Allison' akemiat3...@yahoo.com; Histonet Histonet@lists.utsouthwestern.edu Sent: Monday, April 28, 2014 3:06 PM Subject: RE: [Histonet] Was told I am having bounce-backs I have had the same issue and was told by HistoNet about the problem of bounces. I did get some strange e-mails coming through on HistoNet that I deleted. The problem was they opened in my end mail box due to my view settings before I could get rid of them. I am wondering if this was the problem as I recognized no one on the bounced list I received. I have gotten e-mails today. Pam Marcum -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Akemi Allison Sent: Monday, April 28, 2014 2:59 PM To: Histonet Subject: [Histonet] Was told I am having bounce-backs This is a test to see if this posts on histonet. I was told yesterday that there are a number of bounce-backs. I haven't received emails from histonet the past few days. Anyone else having problems? Peculier issues with yahoo lately Akemi Allison-Tacha, BS, HT (ASCP) HTL Pathology Manager Monterey Bay GI Consultants 23 Upper Ragsdale Drive, Suite 200 Monterey, CA 93940 (381) 375-3577 X117 W. Email: aalli...@montereygi.com P. Email: akem...@yahoo.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Confidentiality Notice: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ 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] Staining FFPE with biotinylated SNA - how to block?
Hi Merissa, don't know if you got any private idea responses so I'll throw in my opinion. I would always worry about some of the things you are mentioning and that are standard thoughts regarding biotin block, retrieval, etc in IHC. But I would think about your serum, which I steadfastly avoided with SNA or any lectin I used. Lectins look at glyco components and serum (or serum substitutes) can be full of glycoproteins and the target then is the blocking serum for your lectin which can cause bad background. I did and would use washes, diluents, etc that had NO serum or milk or anything like that in them. You can make your own, completely free of potentially having glycoproteins or Vector sells some. For some lectins (look at a list of target sugars) you maybe can get by with serum or milk and such to block but many I've found you just can't. Ray (still in, whoever would have guessed, once again rainy Seattle) - Original Message - From: M.O. modz9...@gmail.com To: histonet@lists.utsouthwestern.edu Sent: Tuesday, April 15, 2014 5:59:51 PM Subject: [Histonet] Staining FFPE with biotinylated SNA - how to block? Hello Histonet! I ran a trial on FFPE mouse samples with a biotinylated lectin, SNA from vector. The SNA is Biotinylated Sambucus Nigra Lectin (Elderberry). I have never stained with anything like this, so I ran a test. I deparaffinized, blocked with NGS, incubated overnight at 4C with the diluted biotinylated SNA. On the second day, I used Vector's ABC kit and alkaline phosphatase (red) kit. Once stained, I noticed a lot of background. After looking into the blocking step, would a biotin/avidin blocking step be the correct step instead of a serum because I don't have a secondary? How do I know what needs to be blocked - biotin, avidin or both? Is there a way to do this without a kit and use solutions I may have in my lab? Thank you for your help! Sincerely, Merissa ___ 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] Middle School Science Day and OT diatribe
Hello everyone, thought I'd chime in here as I just returned from helping and judging at the 2-day WSSEF (Washington State Science and Engineering Fair) in Bremerton, Washington. Had 500 incredible projects from all over the state. It is our state fair that leads into the ISEF, giant Intel science fair in May that draws 1,600+ students from all states and 70 countries and territories to compete for prestige, recognition and a million dollars of prizes and scholarship money. So if you have little/no interest in subject heading, please save yourself a bit of reading and just hit delete right now. The data is in and is incontrovertible. The US is getting their teeth kicked in by the world when it comes to hard science and math in education and jobs. At 2-4th grade we are above the other countries of the modern world in science and math. At 5-7 grades, we are at around the same level as other countries and by time of graduating high school, we are down to 25-40th place in science and math amongst those same countries depending on which educational group is testing. So at the elementary and middle school level, I'd encourage all parents and school districts to get more STEM (science, technology, engineering, math) involved before our students start falling off the cliff in STEM education by the end of high school. Michael Titford offers a great suggestion. Staying within the confines of histology/pathology/lab medicine of research these are some of the things I've done in elementary and middle school school either at individual school fairs or my school-district fair. If you can't go full blown with a microscope and such, just having a few paraffin blocks and along with those blocks a cut and stained slide and a photomicrograph, you can reveal some amazing information about lung (being lacy and full of air) or brain or gut (being a tube) or as much as the student can follow along. One thing that I use, rather than a microscope where only one student can look before changing, is one of those old microfiche readers that you can get for a few dollars. Just put a slide in the plate where the microfiche went and see a rather startling enlargement. Yes, is not 1,000x oil immersion resolution but you can see growth plates on appropriately stained bones or bronchi in lung sections or normal skin stained with a melanin stain and a skin with melanoma nodule stained with melanin stain or (fill in as many examples as you want limited only by your imagination and ingenuity). Get a plant stem from garden, process it and cut cx and ls and see tubes or if your student is dissecting a spotted frog, get a piece of skin, fix in isopropyl alcohol for safety, process and on section can see groups of black melanin corresponding to the spots on the gross frog even if you can't see at cellular resolution. The possibilities and permutations for showing such science to a middle-schooler are nearly limitless. And is even great in lab itself to demo faults, folds, chatter, etc. Maybe only downside in real lab is if one of the regulators requires the instrument to go through QA/QC/verification etc. Actually at our WSSEF fair, there were several GREAT projects relating to such things as urothelial carcinoma and IL-38 and a few such subjects and there were some histology micrographs scattered amongst those and some other projects Here is a quote to think about We need to teach our kids that it's not just the winner of the Super Bowl who deserves to be celebrated but the winner of the science fair. When talking about education and jobs now and into the future from Barack Obama in his Jan 25, 2011 State of the Union address. Hope everyone will support STEM, including histology, pathology, lab medicine and lab research at any science fair for any student. Off my soap box. Ray, in suddenly sunny Seattle - Original Message - From: mtitf...@aol.com To: histonet@lists.utsouthwestern.edu Sent: Sunday, April 6, 2014 9:58:17 AM Subject: [Histonet] Middle School Science Day Carol Tanck asks about material for a Middle school science day. Good items to get hold of are plastinated organs that the young people can pick up and handle. If you have a medical school or community college near by, their cellular biology or anatomy departments (Titles vary in these modern times) may be able to lend you some. These organs have been thoroughly fixed, infiltrated with plastic, and then hardened. They are safe to handle and can survive a drop or two. They are especially good for showing lung tissue from smokers showing emphysema, etc. Regards Michael Titford USA Pathology Mobile AL USA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list
Re: [Histonet] RE: Mouse F4/80 antibody
Hello, I agree anti-human CD68 might not be best for mouse tissue but there are specific mouse CD68 (the mouse homolog to human CD68) antibodies out there. FA-11 is one of my favorites. Have used both F4/80 and (rat anti-mouse CD68) on flow, frozens and FFPE. Anna, a literature search gives you published protocols. I'm looking right now at a photo of some CD68+ macs staining on mouse tissues I took from years back. Shows them nicely. anti-mouse CD68 on mouse tissue is in the literature. Ray Seattle - Original Message - From: pru...@ihctech.net To: Anna Hughes anna.c.hug...@gsk.com, histonet@lists.utsouthwestern.edu Sent: Wednesday, February 12, 2014 12:02:07 PM Subject: [Histonet] RE: Mouse F4/80 antibody Rat anti ms F4/80 is used on mouse tissue ffpe for macrophages not anti human cd68. Patsy Ruegg, HT(ASCP)QIHC Ruegg IHC Consulting 40864 E. Arkansas Ave Bennett, CO 80102 H 303-644-4538 C 720-281-5406 prueg...@hotmail.com -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Anna Hughes Sent: Wednesday, February 12, 2014 9:14 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Mouse F4/80 antibody Hi Everyone! I was wondering if any of you have a favorite CD68 macrophage antibody that you have used on mouse tissue and is especially reliable. This is a target that has been notorious in our lab and I was wondering if there was a really good one to use. Thanks in advance for your help! Anna Hughes Anna C. Hughes, BBA, BS, HTLCM anna.c.hug...@gsk.com ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] zebrafish embryos histology
Patty, did some zebrafish work years ago, either pure histology or sections after we did whole mount ISH on the embryo's. As per Jack Ratliff's post, paraffin I found was really tough for orientation and for getting enough sections of such small embryo's. But would use, as Jack suggested, JB-4 Plus and would get beautiful sections, many embryo's in a block and multiple sections per slide. What I would do is under a dissecting scope would, with a VERY fine tool, push the multiple embryo's into an ordered row with similar orientation in the unpolymerized GMA block. Polymerize. If I wanted longitudinal sections, cut the block as is. If we wanted cross-sections, just gross cut the polymerized block and put it in a second GMA block of unpolymerized GMA and stand it up so the embryo's were on end and polymerize . Was every individual embryo correct? No! But enough were so got great HE sections or also seeing the ISH probe revealed at the cellular level. Ray, retired in Seattle - Original Message - From: Patricia F Lott pl...@uab.edu To: histonet@lists.utsouthwestern.edu Sent: Wednesday, January 22, 2014 7:00:40 AM Subject: [Histonet] zebrafish embryos histology Can anyone give me a method for zebra-fish embryo histology? The papers I've read show photos, but no description of histology in M M. I need to put several embryos in each block, and get the orientation correct, and put multiple sections on each slide, in hopes of getting one or two that are perfect. Any suggestions would be greatly appreciated. Thanks, Patty Lott UAB CMBD Core Lab 205-934-2007 ___ 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] zebrafish embryos histology
Hi Patty, You sort of piqued my curiosity of what is going on in the zebra fish world and found this youtube, 6 minute film http://www.youtube.com/watch?v=kZDwo20hl1Efeature=youtu.be About what is going on at Welcome Trust Research on zebra fish but maybe you know all this already. Anyway, I think the film is pretty neat with some incredible (not histology) but CONFOCAL slices through the fish. We had success doing this by confocal on whole-mount but again when studying gene manipulation and cell signal distribution and wanting histology in embryo's, had better luck with GMA. Paraffin as mentioned can certainly be done and is certainly easier but we could never get the resolution or number of sections we desired using paraffin. Maybe the people in lab from this film can give you some suggestions. Ray, retired in Seattle - Original Message - From: koelli...@comcast.net To: Patricia F Lott pl...@uab.edu Cc: histonet@lists.utsouthwestern.edu Sent: Wednesday, January 22, 2014 8:02:24 AM Subject: Re: [Histonet] zebrafish embryos histology Patty, did some zebrafish work years ago, either pure histology or sections after we did whole mount ISH on the embryo's. As per Jack Ratliff's post, paraffin I found was really tough for orientation and for getting enough sections of such small embryo's. But would use, as Jack suggested, JB-4 Plus and would get beautiful sections, many embryo's in a block and multiple sections per slide. What I would do is under a dissecting scope would, with a VERY fine tool, push the multiple embryo's into an ordered row with similar orientation in the unpolymerized GMA block. Polymerize. If I wanted longitudinal sections, cut the block as is. If we wanted cross-sections, just gross cut the polymerized block and put it in a second GMA block of unpolymerized GMA and stand it up so the embryo's were on end and polymerize . Was every individual embryo correct? No! But enough were so got great HE sections or also seeing the ISH probe revealed at the cellular level. Ray, retired in Seattle - Original Message - From: Patricia F Lott pl...@uab.edu To: histonet@lists.utsouthwestern.edu Sent: Wednesday, January 22, 2014 7:00:40 AM Subject: [Histonet] zebrafish embryos histology Can anyone give me a method for zebra-fish embryo histology? The papers I've read show photos, but no description of histology in M M. I need to put several embryos in each block, and get the orientation correct, and put multiple sections on each slide, in hopes of getting one or two that are perfect. Any suggestions would be greatly appreciated. Thanks, Patty Lott UAB CMBD Core Lab 205-934-2007 ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] OT-Retirement
Hello all out there, This is regarding: Ray Koelling; currently from just north of the Seattle, WA area. If you and I have connected in some way over the last 47 years, the following is a message concerning my (semi)-retirement that I hope you will enjoy and anyone with little or no interest in such writing or that knows me not, can easily use the delete button right now to save themselves a few minutes of OT reading. It is time in life to shift a bit of focus after this long and amazing anatomic pathology journey. There was the high school summer job in the mid 60's at a St. Louis histopath lab changing a Lipshaw linear, open air tissue processor (we used dioxane both miscible with aqueous solutions and paraffin and NOT the dioxin of Agent Orange infamy) along with folding A LOT of paper boats for embedding and making 10% formalin from 55 gallon drums of 37% formaldehyde solution. Somehow my brain has survived relatively unscathed. Some may dispute that last sentence. Working at Jackson Memorial Hospital in South Florida with the great Dr. Azorides Morales and Dr. Mehred Nadji (actually Nadji was a resident when I was there and I have a treasured picture of him kicked back at a BBQ at my house wearing his famous sandals). To learning more advanced immunology, histology and a lot more of cellular/molecular techniques along with the ability to critically think during the 5 years (of both unbelievably positive heaven and unrelenting, unforgiving hell) of graduate school. To the biotechnology world and working on such drugs as etanercept, panitumumab, denosumab and (still in testing) TSLP, a compound that I had actually worked on in graduate school but when it was the newly discovered murine form TSLP and then also 50 other discovery molecules that all saw their shelving at various stages of development as failed candidates to progress in a particular pathway. To then being able to work with Dr. Allen Gown in his fantastic lab in the Seattle area. So if we have run into one another, in person, on-line, at a meeting or anywhere in the last nearly half century, hope you are doing well and are keeping safe and I wish you the best. A part of my time now is going to be spent on some K-12 education projects. Organizing and helping at 2 different school districts for district-wide science fairs and STEM (science, technology, engineering, math) career festivals. I am helping at the annual biotechnology fair in the Puget Sound Region for 300-400 high schoolers. And am on Board of the Washington State Science and Engineering Fair that is the entrance point for this state into the big International Intel Science Fair. Why? It is no new, great news flash at all that the US is sinking further behind many countries of the world in math and science education. And that is to the severe, possibly life-long detriment of kids now who will be less able to compete, as adults, with a global economy, jobs and society of the 21st century and which will frankly revolve a lot around STEM issues whether you like it or not. The world is simply not now, nor will ever be again, as it was with me holding a 4-year degree in biology/chemistry in 1973 and at that time having virtually unlimited access to whatever I wanted to do. Then for those kids K-12 who don't like math and science at all and don't want to be in STEM or any kind of STEM career I have offered up this message to them. Not liking STEM as a career is perfectly fine. You need to do in your life what your talents and dreams allow you to do. Yet remember this. No matter what non-STEM thing you do in life, you as a voting, tax-paying, living, breathing adult will be surrounded by STEM issues all throughout life. You will be voting for/against issues or policies and for/against politicians and some, even many of those issues are STEM issues. Radioactive storage waste in salt domes in Nevada? Fracking in the upper Midwest? Coal vs. nuclear vs. green energy anywhere? 5 cent plastic bags to cure global warming? Healthcare? Genetically modified foods? Embryonic vs. other stem cell research? Aging populations? Disease? Mars yes or Mars no? End-of-life issues? Steroids and other drugs in the environment and food stuffs? Sonar testing in oceans? 100 other politically driven STEM-related issues. How do adults now, and then you eventually when older, get your science information? Journalists (on both sides of the political divide) see themselves as having a higher-level moral obligation to now fine-tune and manipulate the news, including science news, instead of just reporting it. Politicians (on both sides of the political divide) spew out any so-called science if it gets them more votes than they loose. Talk show hosts (on both sides of the political divide) spew out science if it gets them more ratings than they loose. Self-promoting, mainly amateurish-science bloggers or
Re: [Histonet] Asbestos microtomy advice?
Hugh, I agree with Rene about the possibilities and yes a mask might be overkill. But far, far, far more than that for me, the description of this research as stated bothers me greatly. I am surely no expert on this particular line of research but it is my understanding that projects looking at mesothelioma and such involve inhalation of asbestos or minute injections into flanks or pleural cavities. No matter the route of administration, if you are UH, your IACUC (Institutional Animal Care and Use Committee) is a federally mandated law governing experimentation on and treatment of animals. Along with several other groups with lettered names. When we or anyone I know does experimentation on animals, they have to absolutely define the limited amount and route of administration and it cannot be altered without prior approval. I could be very wrong, but I can't even imagine any committee following the rules allowing authorization of 75-100 apparently haphazard injections (IP? SQ? ID?) into a mouse much less do this apparently many, many mice. For what purpose? If I was cutting tissues, I wouldn't be so concerned about myself but I would sure want some answers to this exact procedure as you described. Ray Koelling Seattle, WA - Original Message - From: Rene J Buesa rjbu...@yahoo.com To: Hugh Luk hlu...@msn.com, histonet@lists.utsouthwestern.edu Sent: Wednesday, October 9, 2013 7:25:55 AM Subject: Re: [Histonet] Asbestos microtomy advice? If there are so many sites injected and so much injected, during trimming you may expose some fibers and maybe they can float in the air (?), and maybe you can inhale them (?) and maybe you can get asbestosis (doubtful) BUT if you want to be better safe than sorry, use a simple mouth/nose mask. (I personally think this would be an overkill!) René J. From: Hugh Luk hlu...@msn.com To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Tuesday, October 8, 2013 10:09 PM Subject: [Histonet] Asbestos microtomy advice? Hi folks, Odd question (one I never expected until this week): if a researcher was injecting fairly large asbestos loads (~75-100 injections) into research mice, would you use extra precautions while performing microtomy? On thousands of tissue blocks? The injection sites are so numerous, we easily note the colors of the type of asbestos fiber-clusters while grossing. Our concern, is mainly in the act of trimming and microtomizing the tissue. Noteworthy publications for histology? Grateful for any advice (in advance), Hugh UH cancer center Hawaii ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Endogen
I recently had the same question. They were gobbled. Try Endogen antibodies at Thermo Scientific conglomerate. Ray Seattle, WA - Original Message - From: Ronald Houston ronald.hous...@nationwidechildrens.org To: histonet@lists.utsouthwestern.edu Sent: Friday, October 4, 2013 8:16:31 AM Subject: [Histonet] Endogen Does anyone know if Endogen has gone out of business or have they been gobbled up by another larger conglomerate? Trying to get one of their antibodies and can no longer find their web-site Ronnie Houston, MS HT(ASCP)QIHC Anatomic Pathology Manager ChildLab, a Division of Nationwide Children's Hospital www.childlab.com 700 Children's Drive Columbus, OH 43205 (P) 614-722-5450 (F) 614-722-2899 ronald.hous...@nationwidechildrens.orgmailto:ronald.hous...@nationwidechildrens.org www.NationwideChildrens.orghttp://www.nationwidechildrens.org/ ___ 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] Dimwits and off topic
I absolutely agree with well said and I know I'm going to just hate myself in the morning for doing this but I can't stop. I hope everyone (I've mentioned the book to our school board and every kid I mentor in biotechnology and tutor in math) will get The Dumbest Generation:How the digital age stupifies young Americans and jeapordizes our future by Mark Bauerlein, professor of English at Emory University. And although directed at the 20's crowd and under, it sure seems that same mentality is seeping over to the oldsters. The lady who plowed into me last week walking down the hallway totally oblivious and completely immersed in her texting, had to be at least 45 years old. It used to be you had to be rude and uncivil to someones face-to face or behind their back to an individual. Now you can say anything you want, anytime at all, without thinking a bit, to the whole world and often times with anonymity. I agree with the assessment that Rene has good things to offer. 95% of the time and I file those things away. But there are many good people on the HistoNet, they need not be mentioned for people know who they are out there, but even so, not sure any of them have been or should be officially or unofficially elevated to a most respected voice on the Net. As far as histologists (started in 1967 although moved more molecular in the 90's in graduate school) and the respect or disrespect discussions, some of the postings in last couple weeks give credibility to why there might be more disrespect than respect. Is at least some of this stuff that has come through, on a professional forum, much better and more higher level thinking than high schoolers tossing notes back and forth when the teachers back is turned or pulling someone aside to spread the gossip do you know what someone told a friend, who told a friend who told me that. I know I shouldn't but have to hit send and for all the negative e-mail I'll get, I'll read it, won't respond to it but I hope it at least evokes a thought or 2 about civility and humanity that e-mails seem to be taking away. Sent from my desktop computer. Ray in Seattle - Original Message - From: Pamela A Marcum pamar...@uams.edu To: Tim Higgins thiggin...@msn.com, anni...@gmail.com, histonet@lists.utsouthwestern.edu Sent: Friday, September 13, 2013 7:31:33 AM Subject: RE: [Histonet] Dimwits Thank you and well said!! -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tim Higgins Sent: Friday, September 13, 2013 9:17 AM To: anni...@gmail.com; histonet@lists.utsouthwestern.edu Subject: [Histonet] Dimwits Hello Annieinarabia, You are quite the advocate for Sir Rene J. of the most respected voices on this forum. Truly if you want to be respected, give respect and do not degrade other as he does from time to time (in my opinion). Give substance in your responses and not comment using some pompous response to a valid question. Like I said to someone off line, its not that he doesn't have knowledge (so I here, don't know him personally) to answer with useful creditable answers just at time he doesn't. I am sure everyone appreciates his sharing of knowledge, just be constructive in your responses. I don't comment on a subject unless I have useful information to share, never to belittle the person asking who is in search of a solution to a problem. Take that approach and less people will be annoyed with the responses. FYI, there is only one dimwit attached to this response unless your suggesting everyone on Histonet are dimwits. Also, not sure why you are attacking the youngsters on this forum, this forum is for the young, old and middle-aged. Have a fantastic Friday TiminTexas not Tim M. (Don't want anyone mad at him on accident) Message: 13 Date: Fri, 13 Sep 2013 09:52:45 +0400 From: Anne anni...@gmail.com Subject: [Histonet] Re: Histonet Digest, Vol 118, Issue 24 To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: ab6084bc-fab0-44f0-8cc1-0d1c6f141...@gmail.com Content-Type: text/plain; charset=utf-8 My goodness ignorance abounds...Rene J is one of the most respected voices on this forum. And 'she' is actually a 'he' you dimwits. I would take his advice above almost anyone on this forum. You youngsters should try to emulate rather than denigrate. Rene you rock!!! Annieinarabia ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Confidentiality Notice: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is
[Histonet] OT OT OT off topic histologically but still devastatingly sad
Just hours after I sent out my recent e-mail regarding the digital and internet conundrum and how uncivil it can tend to make humans by its indiscriminate use without first any thought, here is a headline copy/paste from the national news: Police: Girl, 12, Bullied Online Before Suicide Is the digital world and the internet the panacea and great wonder that it is purported to be? Are we really as kind and as civil and advanced as humans as we believe we are? Just some non-histologic food for thought. Ray in Seattle ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC after EDTA decalcifying
Could I ask exactly what you are looking for in mouse femur and human tonsil the tonsil I assume is a control? For what? Whatever you are looking for is obviously in human tonsil. Are you positive it is in murine femur. I used to stain for 15-20 targets in EDTA decaled mouse femurs but my control was also a mouse. Ray Research Scientist Comparative Medicine University of Washington - Original Message - From: Colleen Forster cfors...@umn.edu To: Histonet histonet@lists.utsouthwestern.edu Sent: Thursday, August 15, 2013 4:58:35 PM Subject: [Histonet] IHC after EDTA decalcifying Hello fellow histonetters, Can someone who has worked with decalcifyied bone tell me if EDTA interferes with IHC staining? I was under the impression it did not but cannot get staining in mouse femurs that I have decaled in EDTA. I have used both the steamer and the decloaker for retrieval and in the human tonsil the stain is great. Any suggestions.. Thanks in advance! Colleen Forster U of MN ___ 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] another Movats question (paraffin)
Betsy, Don't know if applicable to this situation but in the past I have on occasion in other labs noted some stain going off and after checking everything, looked at the tap water. In fact I backtracked through the water utility once to see where the water came from, was delivered and what the utility did to purify. Convinced, although never proven, that tap water was culprit on at least one or two occasions. Utility would say they used this chemical to purify but on occasion changed to that chemical. Although we fall into the habit that tap water is tap water it can change from time to time (source, purification, metals eroding in old pipes, etc). Don't have to be a chemist to drink tap water in one part of US and find it completely different tasting from another part of country. Maybe not likely cause but worth considering. Ray Research Scientist UW Seattle - Original Message - From: Betsy Molinari bmolin...@texasheart.org To: Histonet@lists.utsouthwestern.edu Sent: Tuesday, May 28, 2013 8:47:19 AM Subject: [Histonet] another Movats question (paraffin) Hi, The Alcian Blue in my Movats is very faint or seems to completely disappear. I have been running Movats for years and this is a new problem. I have checked lot #’s everything is the same. The solution is 1% pH 2.7. The slides are in solution for 20 mins, rinse for 5 mins in tap H2O, then in alkaline alcohol (90 ml 95% 10 ml ammonium hydroxide) for 1 hr then rinsed for 10 mins. But after hematoxylin the blue is either gone or very faint. Any suggestions? Thanks, Betsy Molinari HT(ASCP) Texas Heart Institute Cardiovascular Pathology 6770 Bertner Ave. Houston , TX 77030 832-355-6524 (lab) 832-355-6812 (fax) http://www.texasheart.org Betsy Molinari Senior Histology Research Technician 832-355-6524 | bmolin...@texasheart.orgmailto:bmolin...@texasheart.org | www.texasheart.orghttp://www.texasheart.org 6770 Bertner Ave., MC 1-283, Houston, TX 77030 [Texas Heart Institute][THI News]https://secure3.convio.net/thi/site/SPageNavigator/GlobalSiteOptInPage.html [THI on Facebook] http://www.facebook.com/Texas.Heart.Institute [THI on Flicker] http://www.flickr.com/photos/texasheart/sets/ [THI on Google] https://plus.google.com/u/0/118043615690351997044/posts [THI on Pinterest] http://pinterest.com/texasheartinst/ [THI on Twitter] http://twitter.com/Texas_Heart [THI on You Tube] http://www.youtube.com/TexasHeartInstitute Confidentiality Notice: This message may be confidential and/or privileged. If you are not the intended recipient you may not review, disseminate or copy this e-mail, its contents and/or any attachments. Please immediately notify the sender If you have received this e-mail in error and delete it from your computer system. ___ 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] Safranin O staining
Victor, I completely agree with Tony and Renee's methodology assessment but I would also ask you to look at your induced chondrogenesis model (I've never done this but have done many, many other cell culture models). If you go to this paper http://biotech.korea.ac.kr/bk21/bbs/upload/bk21bbs_cur_165_0.pdf in Tissue Engineering they describe in detail exactly what you are trying to do, if I read your question correctly. Their pictures of safrinin 0 staining of pelleted cells from culture could be described as light staining if you compare them to the red/orange color what we all know safranin o looks like on articular surfaces. I think that is a POOR positive control for their experiments but that's just me. In-vitro cultured cells prepared for histology will seldom stain the same as in-vivo material prepared for histology. And they had to hit cells pretty hard, 5ng TGF to see some staining. Have no idea what your cell culture methodologies are but what I think you are doing is all in that paper and you can see the results in the Safranin o pictures. There are ways to get better, more similar, positive staining controls then they used. Ray Koelling Research Scientist University of Washington Seattle - Original Message - From: Victor Wong vhlw...@yahoo.com To: histonet@lists.utsouthwestern.edu Sent: Sunday, February 17, 2013 6:34:12 PM Subject: [Histonet] Safranin O staining Hi all, I am working on induced chondrogenesis in cell culture. After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block. When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure. I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining. Here is my protocol: 1. Weigert Iron Hx for 2 min 2. Acid alcohol,Scott Tap and wash 3. 0.02% fast green for 2 min 4. 0.1% acetic acid for 1 min 5. Sigma's Safranin O for 5-10 min 6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable. I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose. Are there other factors affect the staining. Any comments are welcomed. Thanks you in advance. Victor ___ 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] anti-GFP staining rabbit polyclonal A11122
Marina, I agree with Paula Pierce, have used the same anti- GFP antibody on some projects but at 1:500-1:1000 area and with a much longer 8-15 minute retrieval in citrate. Also, I know you know to be aware of how much the mice are transfected . In my transfected mice (NOT GFP and in a former life) we might transfect 4 different times and get 4 different efficiencies and 4 different levels of expression in 4 different lines. And of course then in your case the variation with the type of GFP you are transfecting with might ultimately be seen at varying IHC levels since all GFP antibodies don't see all GFP variants with the same efficiency. Something I did to help ease the pain of working up a stain when working up lacZ (not GFP ) was to use a commercially available Tie2/ LacZ transgenic mouse. With that Tie2 promoter driving LacZ expression only in endothelial cells, you had neg tissue (equivalent to wt) and pos endothelial cells (your transgenic target) right in the very same section. It proved to be an exquisitely sensitive control when doing beta-gal studies. Although I've never used it I understand there is now a Tie2/ GFP transgenic available which should help optimize staining protocols as the expression levels are constant and well characterized and you need only one mouse. Don't know what other gene or transgene target you are after but any given transgene does not incorporate equally all the time as you know. Thats my take on this. Ray Koelling PhenoPath Labs Seattle, WA - Original Message - From: Marina Pils Marina. Pils @helmholtz-hzi. de To: histonet @lists. utsouthwestern . edu histonet @lists. utsouthwestern . edu Sent: Monday, February 27, 2012 4:05:56 AM Subject: [ Histonet ] anti- GFP staining rabbit polyclonal A11122 Dear all, We are trying to establish an anti- GFP staining with rabbit polyclonal A11122 from Invitrogen . We are using formalin fixed, paraffin embedded mouse tissue and want to distinguish between a GFP transgenic mouse strain and a wt mouse. The problem is, we get a strong background in the wt, especially in spleen and lymphnodes , while the staining looks fine in liver. We dilute the antibody 1:400 and use 2 min antigen retrieval in Citrate buffer. Can anyone recommend a protocol? Or any other antibody that is working better? Thanks a lot for your help, Marina Marina Pils , DVM , PhD Animal experimental unit Helmholtz Centre for Infection Research Inhoffenstr . 7 D 38124 Braunschweig Helmholtz-Zentrum f?r Infektionsforschung GmbH | Inhoffenstra ?e 7 | 38124 Braunschweig | www .helmholtz-hzi. de Vorsitzende des Aufsichtsrates : MinDir'in B? rbel Brumme-Bothe, Bundesministerium f?r Bildung und Forschung Stellvertreter : R? diger Eichel , Abteilungsleiter Nieders ? chsisches Ministerium f?r Wissenschaft und Kultur Gesch ? ftsf ? hrung : Prof. Dr. Dirk Heinz; Ulf Richter, MBA Gesellschaft mit beschr ? nkter Haftung ( GmbH ) Sitz der Gesellschaft : Braunschweig Handelsregister : Amtsgericht Braunschweig , HRB 477 ___ 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] interview cutting-OT-disarmingly long for deletiondisinterested
Fascinating... and that gets me back to my original ponderance. Why all the false histotechs? Are there people trying to sneak into flow cytometry who have never run a flow cytometer or clinical chemistry medtechs who have never sat in front of an analyzer or cytotechs who don't know an epithelial cell from a glandular cell. What is the reason that there seems to be so much trouble now with non-functioning or poorly functioning histotechs or outright imposters? Maybe there is an answer 2 or 3 levels globally deeper in health care and society than the simple question of Can you cut a section at interview? Ray Seattle - Original Message - From: Angela Bitting akbitt...@geisinger.edu To: Thomas Jasper tjas...@copc.net, Kim Donadio one_angel_sec...@yahoo.com Cc: histonet@lists.utsouthwestern.edu Sent: Tuesday, January 31, 2012 5:36:53 AM Subject: Re: [Histonet] interview cutting-OT-disarmingly long for deletiondisinterested I've had a temp, who we interviewed over the phone, come in and sit down at a microtome and create the most horrendous slides I've ever seen. He lasted a week and we sent him back from whence he came. I don't think he was EVER a Histotech or if he was it was many, many moons ago. Point is.he snowed us all during the interview. Just thought I'd throw that out there. Kim Donadio one_angel_sec...@yahoo.com 1/30/2012 10:01 PM Oh come on. The truth of the matter of why I like to give a manual test to new hires is because people are graduating some Internet programs without the technical skills to function in a lab. Not all. But I've seen a lot. Just saying:) I don't think it should be made a big deal. You take a drivers test to drive. Peoples lives are on the line in each case. Does that a lone mean I don't hire them. Probaly not. I just need to know how much personal investment of my time I am going to need to give .. Runs for her pillow of dreams :). Nite nite Kim Sent from my iPhone On Jan 28, 2012, at 4:25 PM, Thomas Jasper tjas...@copc.net wrote: Ray, Took the time to read your post. You make excellent points. Getting at the gist of your wannabee comments. What boggles my mind is - how or why someone would try to pull something off like that. Sooner or later (hopefully sooner...like before actually hiring them) the charade will be discovered. Misrepresenting oneself and false or misleading information given on an application is generally grounds for dismissal. Seems to me this isn't Leonardo di Caprio and Catch Me If You Can. In the end you are right about finding ways to determine if an applicant is legit. I've come to believe that in the Histology world - if you meet or hear of someone you don't know...someone you do know...knows them. At least that seems to be true almost all the time. Kind regards, Tom Jasper Thomas Jasper HT (ASCP) BAS Histology Supervisor Central Oregon Regional Pathology Services Bend, OR 97701 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of koelli...@comcast.net Sent: Saturday, January 28, 2012 10:23 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] interview cutting-OT-disarmingly long for deletiondisinterested Or as Gayle wisely pointed out it might be interview sectioning to differentiate those who cut out on an interview. While there is no right or wrong to this question, I'm still not convinced that it is a useful tool for you or HR to just have a routine can cut (section) on rotary microtome check box on application the same as you do for a current address or reference contact check box on a form. As I pointed out in my original stupid reply, willfully breaking my own internal rule to avoid taking up these gray (not black and white scientific) discussions, it would depend on the circumstance (unknown person from unknown parts vs. someone from part of the histology community well known). If I call x who I've known for years about an applicant y who is applying and worked with x and am told Oh! y worked for us for last 4 years. He/she along with z and zz were our 3 who sectioned (#) blocks a day. Devastated to see him/her go but know they had to move along with husband/wife. Great cutter and everyone liked him/her. Having him/her sit down to now cut 10 blocks to see if they can cut as a routine question accomplishes WHAT? If someone mysterious with no background walked in, sure have them cut although there have been numerous fantastic options already posted how to weed them out prior to sectioning a finger off. A (purposely) mis-processed block with tissue now shrunken in from block face and a question of we need a recut, what would you do for this block will let you know in about 2 seconds whether or not this is a histotech impostor. Or looking at a blandly stained, necrotic
Re: [Histonet] interview cutting-OT-disarmingly long for deletiondisinterested
Great and fantastically said and to answer your last question in my opinion No!!. My original point when this started. Ray Seattle - Original Message - From: WILLIAM DESALVO wdesalvo@hotmail.com To: koelli...@comcast.net, akbitt...@geisinger.edu Cc: histonet histonet@lists.utsouthwestern.edu Sent: Tuesday, January 31, 2012 7:18:17 AM Subject: RE: [Histonet] interview cutting-OT-disarmingly long for deletiondisinterested I have been following the string and I see the issue from a different perspective. I have always found it difficult to find qualified and registered techs and have been training science degreed individuals as bench techs for several years. I think the issue is identifying the proper individual to add to your team. Technical skill is important, but attitude, aptitude, desire and how they will ultimately fit into and what they will add to the team are by far more important. The question that may be better to ask is How do you cut a section and why do you cut a section?. Just being able to cut a section is not necessarily going to give you enough information to decide if the individual really fits into the team, no better yet, fit into your culture. Hiring an individual that does not fit into your lab/company and can fully support and promote the mission, vision and goals, will not help you. Why this is now becoming more of an issue may be due to the fact that with the shortage of techs in Histology, the situation exists where we have close to full employment of all registered and qualified techs. When that situation occurs, there will be more opportunities for less skill qualified individuals to obtain employment. I would not go so far as to call less skillful techs imposters or false, but maybe book smart and not skill smart. Hiring an individual to perform to the quality and productivity standards of the lab requires significant investment of time to train. Now the catch 22 starts, you must invest time to properly add to your team and you do not believe you have time to invest. Once you bring a new member into your team, there is a cycle (training, functionality and competency) that must take place. You must identify were an individual fits into that cycle and how much time will be required to move them to competency. I have seen both skill qualified and non-skill qualified candidates take the same time to reach functionality. Again, I believe attitude is more key than technical skill. Without the proper attitude you will not quickly reach functionality and functionality allows you to gain time. Competency is the end goal, but functionality is the first critical step. To properly move through the cycle you must have a detailed, documented and functional training process. This whole discussion speaks to me that there is a lack of written and documented standardized training (it works for MT's) and to develop standardized training you must have standardized procedures and techniques (you knew I would work this into the discussion). The degree of difficulty of an individual to meet the quality and productivity requirements of the lab depends on the amount of standardization in the lab. Couple a standardized process with attitude and desire and you can quickly develop a new hire into a fully functioning member of the team. I believe that is the real goal and that will help Histotechnology progress. Is attitude, aptitude and desire really exposed when you ask a candidate can you cut a section? William DeSalvo, B. S., HTL(ASCP) Date: Tue, 31 Jan 2012 14:14:54 + From: koelli...@comcast.net To: akbitt...@geisinger.edu Subject: Re: [Histonet] interview cutting-OT-disarmingly long for deletiondisinterested CC: histonet@lists.utsouthwestern.edu Fascinating... and that gets me back to my original ponderance. Why all the false histotechs? Are there people trying to sneak into flow cytometry who have never run a flow cytometer or clinical chemistry medtechs who have never sat in front of an analyzer or cytotechs who don't know an epithelial cell from a glandular cell. What is the reason that there seems to be so much trouble now with non-functioning or poorly functioning histotechs or outright imposters? Maybe there is an answer 2 or 3 levels globally deeper in health care and society than the simple question of Can you cut a section at interview? Ray Seattle - Original Message - From: Angela Bitting akbitt...@geisinger.edu To: Thomas Jasper tjas...@copc.net, Kim Donadio one_angel_sec...@yahoo.com Cc: histonet@lists.utsouthwestern.edu Sent: Tuesday, January 31, 2012 5:36:53 AM Subject: Re: [Histonet] interview cutting-OT-disarmingly long for deletiondisinterested I've had a temp, who we interviewed over the phone, come in and sit down at a microtome and create the most horrendous slides I've ever seen. He lasted a week and we sent him back from
Re: [Histonet] Interviewing Histotechs...
I hope there is at least a bit more to clinical histology than quantity and speed. Maybe I know the particular high schooler you mentioned who was so good since I mentored that Mercer program/class for years and years and even brought in class-wide IHC hands-on projects. Before the teacher left and the program collapsed. Ray Seattle, WA - Original Message - From: Jerry Ricks rosenfeld...@hotmail.com To: histonet@lists.utsouthwestern.edu Sent: Monday, January 30, 2012 11:13:11 AM Subject: [Histonet] Interviewing Histotechs... I gather it is different in clinical labs than in research labs. In clinical labs there is an emphasis on quantity and speed. In research the emphasis is on doing good experiments. Our patients are almost always deceased or shortly about to be so there is no urgency of diagnosis factor. For us, diagnosis means making precise measurements else some scientists looking at an image and asking each other what the? Anyway I always assume that the person I am hiring is incompetent at histology and that they will need to be personally trained by me. Doesn't matter how much experience they have. And over 23 years that has turned out to be true. I've met exactly two people who didn't need much training. One was a former senior clinical lab manager. The other was a kid straight out of high school who happened to have a histology experience from high school and a decent histo portfolio. Yes, Mercer Island High School had a Histology program. No such thing as a tech who doesn't need to be trained and any tech trained by me will be up and running in a week or two. Why bother making them cut or stain anything during a darn interview. If they are smart and cooperative they will work out. If I ever go to a new lab with a new microtome, new protocols, I am pretty sure that I will be sort of incompetent for a week or two as well. Jerry Ricks Research Scientist University of Washington Department of Pathology histonet@lists.utsouthwestern.edu Date: Sun, 29 Jan 2012 13:12:09 -0500 From: rsrichm...@gmail.com To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Re: interview Ray Koelling asked me: If the Samurai Pathologist is out there reading still; any idea over your career, about how many glass slides have you viewed under a microscope since the first? Your replies are always top-notch, entertaining and informative. And hope with each new job you don't have to show someone you can pass a test of which slide shows normal liver and which slide shows cirrhotic liver in your interview. I really have no idea how many slides. In a normal year I sign out about 3,000 histology cases (remember I don't work full time) averaging maybe 3 slides per case. Generally I've gotten jobs, both private clients and agency clients, by recommendation. A number of years ago I was interviewed by a four-pathologist hospital group who handed me a tray of 20 slides with the necessary historical information, and was told that this was a set the group had collected, including very straightforward cases, cases with serious diagnostic pitfalls, and some cases they'd never been able to make a diagnosis on. They tried to make it a test of judgment rather than simple diagnostic skill. Told to take as much time as I needed. I guess I passed - by coincidence, the entire group chanced to break up very quickly, and an entirely different team took over. Bob Richmond Samurai Pathologist Knoxville TN ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] interview cutting-OT-disarmingly long for deletion disinterested
Or as Gayle wisely pointed out it might be interview sectioning to differentiate those who cut out on an interview. While there is no right or wrong to this question, I'm still not convinced that it is a useful tool for you or HR to just have a routine can cut (section) on rotary microtome check box on application the same as you do for a current address or reference contact check box on a form. As I pointed out in my original stupid reply, willfully breaking my own internal rule to avoid taking up these gray (not black and white scientific) discussions, it would depend on the circumstance (unknown person from unknown parts vs. someone from part of the histology community well known). If I call x who I've known for years about an applicant y who is applying and worked with x and am told Oh! y worked for us for last 4 years. He/she along with z and zz were our 3 who sectioned (#) blocks a day. Devastated to see him/her go but know they had to move along with husband/wife. Great cutter and everyone liked him/her. Having him/her sit down to now cut 10 blocks to see if they can cut as a routine question accomplishes WHAT? If someone mysterious with no background walked in, sure have them cut although there have been numerous fantastic options already posted how to weed them out prior to sectioning a finger off. A (purposely) mis-processed block with tissue now shrunken in from block face and a question of we need a recut, what would you do for this block will let you know in about 2 seconds whether or not this is a histotech impostor. Or looking at a blandly stained, necrotic section under microscope and asking interpret this section will tell you something of who or what this person is. Personally, I'd far rather have a person who is energetic, scientifically and intellectually confident and talented, personable, works well within the symphony of histology and cuts 8 blocks and leaves a few wrinkles in this new environment set-up than a (female or male) diva who cuts 10 perfect blocks but who has that nearly imperceptible tint of not a complete team player or dubious personality. A routine check box can cut I think is just a waste of time and resources unless a particular circumstance warrants it. Someone asked would you hire a secretary without a wpm typing test. Absolutely, beyond any doubt. If the transcriptionist next door wants a secretary position and routinely types 3 times faster than is required as a secretary; why a wpm test? If I call someone I know across state where this applicant worked for last 10 years and she's an immaculate and fast typist beyond anything we've ever had and so sorry she had to move, I'd rather then concentrate on more esoteric matrices than wpm. If he/she was a secretary 25 years ago and has been a house-husband or house-wife for 25 years and starting back now or if someone walks in off the street to apply then beyond any doubt; they take a typing test. Someone pointed out that all musicians play their instrument in application to test for the orchestra. Of course but for a completely different reason. You could give an oral test to 1,000 musicians of which 999 would know how to transpose 3 pitches up by 7 semi-tones or define a diatonic scale or identify the composer if listening to an excerpt from the Overture-Midsummers Night Dream. That's not what the interviewee is looking for. They are looking for the ONE in 1,000 who has the exact pitch, timbre, affannato, vibrato, arioso and legato from their specific instrument that only that particular person's instrument and ability possesses. Only a finely trained ear (the conductor) has that God-given ability of relative/perfect pitch or undefinable gift to identify that one instrument and one ability to fit into the total music experience. And there is only one way to find out; have him or her play. Totally different scenario than in a histology lab unless the object is to see how well the speed and noise of one person's cutting blends in with the symphony of 75 other microtomes being used in the lab at the same time. Then you start to ponder, as did a fine mind out there who understood the butterfly comment, if a current 30-year superstar of histology walked into a lab looking for a histology job, would they take a cutting ( sectioning?) test? If Yo-Yo Ma or James Galway or Itzhak Perlman or John Cerminaro had ever walked in to test for an orchestral position, surely they wouldn't be tested just to see if can they play a cello or flute or violin or French Horn or even how well they play on that particular day in that particular environment. Maybe what I'm mis-understanding is that apparently there are A LOT of histology wannabees, walking in off the street trying to sneak into histology? and if so that seems like there should be some manner of response to that situation although not sure what it is. But something short of sitting down to cut
Re: [Histonet] Interview Questions
This is certainly an interesting thread and I generally hate to get into these ever but I still can't figure out one thing and never have over all these years in pathology. What other endeavor in life and job seeking is an on-the-spot demo that you can do something required at a job interview? Does a lawyer have to go into a courtroom for 5 minutes and show he/she can say I object? Does a sanitation worker have to go round the block once and show he/she can empty 9 cans in 5 minutes? Does a doctor need to show he/she can use a stethoscope? Does a bricklayer have to show he/she can lay 20 bricks in 2 minutes? Or fail the interview? Does a med tech have to show they can stain 6 tubes with CD4 and CD 8 and successfully put them on a flow cytometer? Does an actuary have to show they can really add 100 4-digit numbers on a calculator without a mistake? Does a grocery bagger boy /girl have to show they can put x number of items in 3 bags? Does a Pathologist have to show they know how to turn on a microscope and look through it? Does a peanut counter have to show they can count peanuts? I just can't get into my mind the necessity of someone having to cut to show they can cut? What other profession does this at an interview? Now certainly you can come up with scenarios where it might be important to find out. A brand new histotech whose only cut 3 blocks in their life. A tech from the deepest, darkest nether regions of the earth where you cannot check on their background. But a tech whose has been working cutting the last 3 or 7 or 15 years and you've verified with a previous company that is exactly what they did; how will them cutting for 10 minutes further stratify them into yes or no categories. If 2 potential techs cut and one finishes in 9 minutes and one in 10 minutes, is that a true qualifier or disqualifier of what they can do cutting? There are a myriad of things I'd love to know and always ask; personality, job knowledge, wants, desires, needs, ambitions, etc, etc, etc. My blood pressure skyrockets when I give blood because I HATE anyone sticking a needle in me. But I have a really needed blood type. Should nervousness each time disqualify me. This still boggles my mind about what is being accomplished with cutting during an interview? Ray Seattle, WA - Original Message - From: joelle weaver joellewea...@hotmail.com To: trathbo...@somerset-healthcare.com, billodonn...@catholichealth.net, sbree...@nmda.nmsu.edu, Histonet histonet@lists.utsouthwestern.edu Sent: Wednesday, January 25, 2012 10:02:39 AM Subject: RE: [Histonet] Interview Questions Love this! I always want to do demonstration during technical interviews, but usually get shot down from managers and argued with in general, as in people don't feel that they should have to prove they can do histology. This perception, I never got, because I always saw it as in a job interview-in what other situation are you more trying to prove or impress with your knowledge, attitude, skills and experience? If you do bench work, you can tell in just a few minutes of observation much more information than you could get with quite a few questions. To be fair, I take into account nervousness, being closely observed, and lack of familiarity with equipment etc. I don't know, I think its fair if those are important skills to the position/role. Was not sure if Sara's job was mostly technical though, so thought I might keep it general. Joelle Weaver MAOM, (HTL) ASCP http://www.linkedin.com/in/joelleweaver From: trathbo...@somerset-healthcare.com To: billodonn...@catholichealth.net; sbree...@nmda.nmsu.edu; histonet@lists.utsouthwestern.edu Date: Wed, 25 Jan 2012 17:47:01 + Subject: RE: [Histonet] Interview Questions CC: If your replacement will be doing actual histology, will your institution permit the applicant to embed and cut? Can you sit down at a multi-head scope and review slides with them? What will the person be responsible for? Do they have experience with all of these tasks? What would they do in a crisis situation (you can make up one yourself that would be plausible). People who volunteer in their personal lives, may do the same at work. Ask how they juggle their schedule though, if there is a lot going on in their personal lives. Be careful with how you ask these questions though. Your HR department should be able to give you guidance in how to phrase things. Good luck. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of O'Donnell, Bill Sent: Wednesday, January 25, 2012 12:19 PM To: Breeden, Sara; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Interview Questions It would seem that questions like How do you feel about cannibalism? might also be out but might be far more helpful; than phone questions. On the serious side, when I was
Re: [Histonet] Interview Questions
Not upset in the least. Just posting my own questions and doubts within the parameters of the situation. When the Chinese philosopher who fell asleep under a tree and dreamt he was a butterfly and then spent the rest of his life asking if he was a human who fell asleep under a tree and dreamt he was a butterfly or was really a butterfly dreaming he was a human who fell asleep under a tree who? Wouldn't say he at all took offense to the situation; pondering, reflecting and just asking a question. Ray Seattle Sent from my Bedroom Wireless Laptop - Original Message - From: joelle weaver joellewea...@hotmail.com To: koelli...@comcast.net Sent: Wednesday, January 25, 2012 7:26:37 PM Subject: Re: [Histonet] Interview Questions Well I am sorry that you took such offense, but some jobs do have say words/minute typing for example. I guess the variation in qualified individuals leads me to not be upset to be asked to demonstrate tasks within the assigned duties. I think maybe you have simplifed a bit too. I think all those professions,such as attorneys have to do much more than you indicate_sorry this upset you Sent from my Verizon Wireless BlackBerry -Original Message- From: koelli...@comcast.net Date: Thu, 26 Jan 2012 02:59:49 To: joellewea...@hotmail.com Cc: trathbo...@somerset-healthcare.com; billodonn...@catholichealth.net; sbree...@nmda.nmsu.edu; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Interview Questions This is certainly an interesting thread and I generally hate to get into these ever but I still can't figure out one thing and never have over all these years in pathology. What other endeavor in life and job seeking is an on-the-spot demo that you can do something required at a job interview? Does a lawyer have to go into a courtroom for 5 minutes and show he/she can say I object? Does a sanitation worker have to go round the block once and show he/she can empty 9 cans in 5 minutes? Does a doctor need to show he/she can use a stethoscope? Does a bricklayer have to show he/she can lay 20 bricks in 2 minutes? Or fail the interview? Does a med tech have to show they can stain 6 tubes with CD4 and CD 8 and successfully put them on a flow cytometer? Does an actuary have to show they can really add 100 4-digit numbers on a calculator without a mistake? Does a grocery bagger boy /girl have to show they can put x number of items in 3 bags? Does a Pathologist have to show they know how to turn on a microscope and look through it? Does a peanut counter have to show they can count peanuts? I just can't get into my mind the necessity of someone having to cut to show they can cut? What other profession does this at an interview? Now certainly you can come up with scenarios where it might be important to find out. A brand new histotech whose only cut 3 blocks in their life. A tech from the deepest, darkest nether regions of the earth where you cannot check on their background. But a tech whose has been working cutting the last 3 or 7 or 15 years and you've verified with a previous company that is exactly what they did; how will them cutting for 10 minutes further stratify them into yes or no categories. If 2 potential techs cut and one finishes in 9 minutes and one in 10 minutes, is that a true qualifier or disqualifier of what they can do cutting? There are a myriad of things I'd love to know and always ask; personality, job knowledge, wants, desires, needs, ambitions, etc, etc, etc. My blood pressure skyrockets when I give blood because I HATE anyone sticking a needle in me. But I have a really needed blood type. Should nervousness each time disqualify me. This still boggles my mind about what is being accomplished with cutting during an interview? Ray Seattle, WA From: joelle weaver joellewea...@hotmail.com To: trathbo...@somerset-healthcare.com, billodonn...@catholichealth.net, sbree...@nmda.nmsu.edu, Histonet histonet@lists.utsouthwestern.edu Sent: Wednesday, January 25, 2012 10:02:39 AM Subject: RE: [Histonet] Interview Questions Love this! I always want to do demonstration during technical interviews, but usually get shot down from managers and argued with in general, as in people don't feel that they should have to prove they can do histology. This perception, I never got, because I always saw it as in a job interview-in what other situation are you more trying to prove or impress with your knowledge, attitude, skills and experience? If you do bench work, you can tell in just a few minutes of observation much more information than you could get with quite a few questions. To be fair, I take into account nervousness, being closely observed, and lack of familiarity with equipment etc. I don't know, I think its fair if those are important skills to the position/role. Was not sure if Sara's job was mostly technical though, so thought I might keep it general. Joelle
Re: [Histonet] RE: B-cell Ab to work on rat tissue
Carl's choice is excellent. The classical B-cell marker might be CD20, and does work great, but as with all antibodies, you have to be aware of exactly what you are after. CD20 comes on line after CD19 induction so CD20 might not be seen on very early b-cells. Like wise CD79a is accompanied by chaperone proteins prior to it's assemblage with the B cell receptor (during the pro-B stage). Won't mark these very early cells well. The assemblage is complete at pre-B stage and continues expression even till plasma cell stage. So true for almost all b cells, CD79a is great and you can see rat tissue IHC of CD79A out on the web, have used several of them, along with protocols (HIER as Carl pointed out works well). The complex is found on virtually no other cell as it is the signalling complex for the b cell receptor. Ray Ray Koelling PhenoPath Labs Seattle, WA - Original Message - From: Carl Hobbs carl.ho...@kcl.ac.uk To: histonet@lists.utsouthwestern.edu Sent: Sunday, January 15, 2012 8:24:59 AM Subject: [Histonet] RE: B-cell Ab to work on rat tissue It may be that CD79a Ab is appropriate? It works well on Pwax sections, after HIER. I do not know the specificity of this Ag for B cells, so would be interested in feedback Check here for images : http://www.immunoportal.com/modules.php?name=gallery2 Carl Hobbs Histology Manager Wolfson CARD School of Biomedical Sciences Kings College London Guys Campus SE1 1UL Tel: 020 78486813 Fax: 020 78486816 020 78486813 ___ 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] help
Hi Lydia, I think Tony Henwood has it exactly right in talking of DAB intensification. The article he sites and several others show how much more sensitive DAB intensification can make an ordinary iron reaction. And you are not looking in bone marrow or spleen but somewhere where there is so little iron. More than once I've been burned by research colleagues who gave me tissue saying x should be upregulated and spend weeks looking for it until they say oop's, sorry-my bad. Intra-dermal injections can become sub-dermal. IP injections can be sub-optimal with rough handling of mice. IV tail vein injections can be missed. And MPTP is toxic and dangerous enough to work with that I'd be fumbling around and nervous myself. If you are confident the model is working by some secondary marker such as tyrosine hydroxylase immunoreactivity then you are still are looking for minute quantities of iron. Several easily available papers on that very mouse model tell you the limited cell population to look for (one says-NOT in the big cells), and in a very specific region using coronal sections (I always used a mouse brain mold to section to be sure of the anatomical location) and several of the papers use classical iron histochemistry followed by DAB intensification and their procedures are in material and methods. Reaction might be working after all-just have to focus in on very limited reaction product. Good luck hunting. Ray Ray Koelling PhenoPath Labs Seattle, WA - Original Message - From: Lydia Gunawan lydia.guna...@unimelb.edu.au To: Histonet@lists.utsouthwestern.edu Sent: Tuesday, November 15, 2011 2:00:32 PM Subject: [Histonet] help Hi there, I am having trouble with Turnbull staining. Anybody can help me? As I want to detect and quantify iron in the brain for Parkinson experiment, I am using mice(C57Bl6) brain that were treated with MPTP injection. So, I have been trying to stain those brain using paraffin section with Turnbull blue but I have no luck. FYI, I have been using 7% of Potassium ferricyanide in 3% HCL and I incubated for 2 hours, 37'. I also did incubation in triton-x and H2O2 too but still not getting any iron on my sections. Could anyone help me to solve my problem? Thanks Lydia Gunawan Oxidation Biology Laboratory Mental Health Research Institute Melbourne Brain Centre Corner Royal Pde and Genetics Lane University of Melbourne, Level 4 Parkville, Vic 3010 email: lydia.guna...@unimelb.edu.au ___ 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] EM question
Cheryl, Have done some things with ordinary sectioning cryo-electron microscopy but not your specific application. But can go to Cold Harbor Springs Protocols and they can get Immersion Freezing of Cell Monolayers for Cryo-Electron Tomongraphy. Even better (more immediate) go here http://www.jove.com/video/1943/electron-cryotomography-of-bacterial-cells and is a California Institute of Technology (really well produced) video/audio/written explaination of the whole procedure. Ray Ray Koelling PhenoPath Labs Seattle, WA - Original Message - From: Cheryl Crowder ccrow...@vetmed.lsu.edu To: histonet@lists.utsouthwestern.edu Sent: Monday, November 14, 2011 4:20:35 PM Subject: [Histonet] EM question I am the first to admit I know little to nothing about EM. I have a researcher who read an article today that said in the method that they had taken cells, applied them to copper EM grids and immersed them in liquid nitrogen to fix them. Has anyone ever heard of this technique? and if you have, do you have any directions or references for this technique? Cheryl ___ 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] Flk2 / Flt3 / CD135 on mouse bone
Adam/his fellow grad student, I could be somewhat off not being up to date with current literature. Sorry. But used to look for Flk2/Flt3/CD135 in murine bone marrow and not the bone itself. In immature hematopoeitic progenitor cells. Along with looking in thymus, etc. But for something like femurs, I removed the bone marrow, and there are some slick ways to do it without disrupting the core architecture too much. Could take it out and obviously flow (cytometry) the cells. But could also get out a fairly intact core and freeze it, or paraffin process/section it or glycol methacrylate section it and worry very little about the minute amount of trabecular bone in murine bone marrow. Depending on age, etc of course. So unless for the specific needs of the project call for looking actually for staining within the cortical bone, I'd take that problem out of the picture by getting the bone marrow out and then not having to worry about decalcification and tape transfer and things like that. Makes IHC staining a lot easier in my experience but perhaps there are those out there who have worked out whole, intact mouse bone CD135 staining. Ray Ray Koelling PhenoPath Labs Seattle WA - Original Message - From: Adam . anonwu...@gmail.com To: histonet@lists.utsouthwestern.edu Sent: Monday, June 27, 2011 5:49:23 PM Subject: [Histonet] Flk2 / Flt3 / CD135 on mouse bone Hi all, One of my fellow graduate students is trying to perform immunofluorescence on PFA fixed, EDTA decalcified mouse bone for Flk2 (aka Flt3 or CD135). So far, he hasn't had any luck on either paraffin embedded or frozen sections using HIER. Has anyone done this successfully? I know the ideal way to perform IF on bone is using a tape-transfer system, but, alas, we don't quite have that working yet. Thanks, Adam ___ 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] Where can Fast Blue (Diamidino compound 253/50) be obtained?
Hello Per, Have no particular interest in neuronal retrograde tracing and have certainly never used anything for that. Just general scientific curiosity and a few minutes time awaiting a home project. Found Fast Blue (synonym: Diamidino compound 253/50) on Sigma website cat # F5756. Good luck. Ray Ray Koelling PhenoPath Labs Seattle WA - Original Message - From: Per Magne Knutsen pknut...@physics.ucsd.edu To: histonet@lists.utsouthwestern.edu Sent: Friday, February 4, 2011 11:22:41 PM Subject: [Histonet] Where can Fast Blue (Diamidino compound 253/50) beobtained? Hi, I've searched every catalog and website for Fast Blue (Diamidino compound 253/50), widely used for neuronal retrograde tracing. One recent paper using this compound is: Porreroa et al Mapping of fluorescent protein-expressing neurons and axon pathways in adult and developing Thy1-eYFP-H transgenic mice, Brain Research, 1345, 59-72 where the authors as many before them cite Dr. Illing GmbH Co. KG, Groβ-Umstadt, Germany as the origin. I have been unable to locate this particular vendor. I've written the authors and am waiting for an answer. In the meanwhile, can anyone on this list help? Kindly, *Per M Knutsen *Department of Physics University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0374 T: +1 858 405 2868 E: pknut...@ucsd.edu W: http://pmknutsen.blogspot.com * * ___ 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] HE Stain
Allison/Toni, Thought I'd throw this out. Maybe nonsense. If you have such acidic tap water, could there be heavy metals lead, magnesium and others (acid tap water does that) in your tap water rinse from being leached out upstream. William DeSalvo talked about the quality of tap water fluctuating. Very true. And the metals from pipes or solder , leached into water by pH6.0, turning a normal hematoxylin into something like a Weigerts hematoxylin. A kind of post-mordanting that I think some call afterchroming. Although if you tried distilled or deionized water with same results, that data wouldn't fit with this problem. And even if it just started happening, has someone recently worked on pipes upsteam of where you are and there is (are) new metals being leached into your hematoxylin rinse? pH 6 is pretty acidic water. RayKoelling PhenoPath Labs Seattle, WA - Original Message - From: Toni Rathborne trathbo...@somerset-healthcare.com To: WILLIAM DESALVO wdesalvo@hotmail.com, jkier...@uwo.ca, allison scott allison_sc...@hchd.tmc.edu Cc: histonet histonet@lists.utsouthwestern.edu Sent: Thursday, January 20, 2011 6:17:46 AM Subject: RE: [Histonet] HE Stain Our tap water consistently reads 6.0, and has for years. We did try turning off the tap when this first began, and manually rinsing with distilled water, but saw no difference. I will try adding the HCl today with a few test slides. Will let you know how this works out. Thanks for the suggestions. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu]On Behalf Of WILLIAM DESALVO Sent: Thursday, January 20, 2011 12:26 AM To: jkier...@uwo.ca; allison_sc...@hchd.tmc.edu Cc: histonet Subject: RE: [Histonet] HE Stain I agree that the pH might be high, but I also suggest you check your water rinse on the stainer. If you are using tap water, there can be a significant fluctuation in the quality of the water and the amount of additives and impurities present at any one time can also contribute to the mucin not being rinsed away and staining. If you are using tap water, changing to distilled or dionized water might help to improve the consistency of stain results. Good luck William DeSalvo, B.S., HTL(ASCP) From: jkier...@uwo.ca To: allison_sc...@hchd.tmc.edu Date: Wed, 19 Jan 2011 16:58:57 -0500 Subject: Re: [Histonet] HE Stain CC: histonet@lists.utsouthwestern.edu Sounds as if the pH of your haemalum is too high. Try adding a little HCl to bring it down to slightly above 2. Check a few slides without eosin counterstaining. Nuclei should be blue with very little else stained. John Kiernan Anatomy Cell Biology University of Western Ontario London, Canada = = = - Original Message - From: Scott, Allison D allison_sc...@hchd.tmc.edu Date: Wednesday, January 19, 2011 13:01 Subject: [Histonet] HE Stain To: histonet@lists.utsouthwestern.edu Hello to all in histoland and Happy New Year. We are having issues with our HE stain. The nuclei are staining very blue to purple and the mucin is staining blue to purple-blue. It is difficult to see the nuclear detail. The mucin is obscuring things. We have not changed our process for staining or processing. The funny thing is that it is only in the Biopsy cases, and it is every few slides. The surgical cases are all right. We checked the alcohol and xylene for water, and there is not any. My tech changed out the stain and we are staining a new batch of slides. If anyone has any idea what is wrong, any help would be greatly appreciated. I have gone over our processes and nothing has changed. The reagents are the same, the staining times are the same, and the processing times are the same. We are using the Shandon Gemini stainer and VIP processor. Allison Scott HT(ASCP) Histology Supervisor LBJ Hospital Houston, Texas CONFIDENTIALITY NOTICE: If you have received this e-mail in error, please immediately notify the sender by return e-mail and delete this e-mail and any attachments from your computer system. To the extent the information in this e-mail and any attachments contain protected health information as defined by the Health Insurance Portability and Accountability Act of 1996 (HIPAA), PL 104-191; 45 CFR Parts 160 and 164; or Chapter 181, Texas Health and Safety Code, it is confidential and/or privileged. This e-mail may also be confidential and/or privileged under Texas law. The e-mail is for the use of only the individual or entity named above. If you are not the intended recipient, or any authorized representative of the intended recipient, you are hereby notified that any review, dissemination or copying of this e-mail and its attachments is strictly prohibited.
Re: [Histonet] CD20 for IHC on mouse FFPE tissue
Hello, Maybe I'm remembering wrong since I deleted the original message of Bretts looking for murine CD20 on mouse FFPE (hopefully rabbit origin?). If so anti-murine CD20 that can be made to work on mouse FFPE tissues certainly exist. Have used them from BD (flow reagent), Santa Cruz and others. In Biocompare website, can see them plus new rabbit monoclonal anti-murine CD20 for paraffin, reactive to murine tissues. There are a few CD20+ murine B-cells that have no B220, and vice-versa so I would'nt look for CD45R if I wanted to find CD20+ cells. Would look for CD20+ cells. Maybe I'm remembering original post incorrectly. Ray Ray Koelling PhenoPath Labs Seattle, WA - Original Message - From: Amos Brooks amosbro...@gmail.com To: brett connolly brett_conno...@merck.com, histonet@lists.utsouthwestern.edu Sent: Thursday, November 25, 2010 3:31:27 AM Subject: [Histonet] CD20 for IHC on mouse FFPE tissue Hi Brett, CD20 is not going to work for mouse tissue. You need CD45R (clone B220). This will selectively label B cells just like CD20 in humans. I get this from BD Biosciences (Now Thermo ... again) Catalog # 347460. It is raised in Rats and works great as long as you have a good rat secondary. The one from Jackson is good. Happy Thanksgiving, Amos ___ 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] how to make crashed ice?
Hello, It is very inexpensive, a few dollars, to buy a counter-top ice crusher, that a professional bartender might use to crush ice for drinks. Can be bought at most any store. We crush ice every day on a daily basis to create a small tub of ice we need for reagents. Only the size of like a small kitchen food processor. Just add ice cubes and crush them in a few seconds without a hammer and the mess. Ray PhenoPath Labs Seattle, WA - Original Message - From: louise renton louise.ren...@gmail.com To: gu lang gu.l...@gmx.at, Histonet@lists.utsouthwestern.edu Sent: Saturday, August 7, 2010 3:24:17 AM Subject: Re: [Histonet] how to make crashed ice? Hi gudrun, Place the ice - ice cubes works best in a plastic bag, wrap in a towel and bash with a heavy object like a hammer. You can also use Jamie Oliver's trick - put ice cubes in a cloth tea towel, , bring the 4 corners together, tie them in knot, and then hit the parcel on the edge of your work surface until the ice is the sze you need hope this helps On Sat, Aug 7, 2010 at 12:12 PM, Gudrun Lang gu.l...@gmx.at wrote: Hi! I think this is a rather basic question ;-), but I'm looking for practical advice. We are going to try the OSNA-test for sentinel nodes. The application needs a pot with crashed ice while desintegrating the tissue with a mixer. So over the day this should be four to six litre ice, if we have to take fresh ice for each time, a group of sentinels has to be worked up. What is a practical way to make crashed ice in the lab? Thanks for your answers in advance! Gudrun ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Louise Renton Bone Research Unit University of the Witwatersrand Johannesburg South Africa +27 11 717 2298 (tel fax) 073 5574456 (emergencies only) There are nights when the wolves are silent and only the moon howls. George Carlin No trees were killed in the sending of this message. However, many electrons were terribly inconvenienced. ___ 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] To detect injected mouse antibody inside mouse tissue
Amy, In my opinion the answer is, Yes, certainly possible. Also in my opinion the answer is No, probably impossible. Even with a Yahoo address I'm assuming that this is a research/biotech model and project. In a previous life, when quietly gagged, I went about this on numerable occassions. Sometimes extraordinarily successfully and sometimes complete failures. Done at both light IHC level and with EM IHC. Depends on your specific antibody molecule, how many target receptors on a given cell, pharmacokinetics of the injected antibody, making sure it gets to target and in sufficient quantity, not sequestered inappropriately, how long animal sacrificed post-injection. Probably 5 other variables, each with nuances so just impossible to answer generally in this venue for your specific problem. There are several papers on the subect if you look them up but they are specific procedures for a very specific model. If successful, results are spectacular. More often than not, you spend months on something that in retrospect was probably not doable in the first place after you sit and really think it through. I once got bitten to the tune of several months work and at the end we found out something about the glycosylation of the antibody that would never allow us to localize it appropriately so we had been searching for something we could never find by IHC. Ray Raymond Koelling PhenoPath Labs Seattle, WA - Original Message - From: Amy Lee amylee...@yahoo.com To: histonet histonet@lists.utsouthwestern.edu Sent: Thursday, January 21, 2010 2:19:35 PM GMT -08:00 US/Canada Pacific Subject: [Histonet] To detect injected mouse antibody inside mouse tissue Hello, I have a question regarding detecting injected mouse antibody in mouse tissue by IHC. Now I have mouse liver paraffin section. This mouse was injected mouse antibody. I was asked to locate this antibody. This is a mouse chimeric antibody. Variable regions from human antibody was combined with mouse IgG2a heavy chain and kappa light chain constant regions. I am thinking using rabbit anti-human IgG, F(ab) fragment specific. Do you think it's doable or you have any good suggestion? When this mouse antibody bind to antigen inside liver through this human variable region, my F(ab) fragment specific antibody can still bind to it? Hope I describ my questions clearly. Thank you in advance for any help! Amy ___ 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 Zinc Fixative supplier/source
Hi Gayle, I've used this extensively. But never from BD. Was too simple to make in house and then we had control over tweaking it if needed. Made it up per the formula you wrote and with all your caveats and warnings with which I completely agree. We loved it for certain things. For instance, was NOT good on tissues that were loaded with digestive enzymes-like pancreas. Pancreatic tissue looked like road kill although islet of L cells looked and stained good. Although given the moniker of fixative it certainly isn't..in the classical sense of a fixative. And as you alluded to it is available in Europe. One of our post-docs worked with the company when in Europe. But expensive beyond reason. So we just made it. Ray Raymond Koelling PhenoPath Labs Seattle, WA - Original Message - From: gayle callis gayle.cal...@bresnan.net To: histonet@lists.utsouthwestern.edu Cc: koelli...@comcast.net Sent: Thursday, October 15, 2009 9:03:01 AM GMT -08:00 US/Canada Pacific Subject: [Histonet] IHC Zinc Fixative supplier/source Concerning IHC zinc fixative supplier/source FIRST, Z-Fix from Anatech is NOT the formalin free Beckstead Zinc fixative (BD Pharmingen) commonly used for rodent and human CD/leukocyte marker work. Kathy wrote: I am searching for a vendor to supply IHC Zinc fixative. Not zinc formalin. It's ALMOST like the BD Pharmingen IHC zinc fix, but contains Zinc acetate too. We do not want to make it up in house thoughit contains calcium acetate, zinc acetate, zinc chloride and tris buffer. I have searched the 'net and all I garnered was a head ache, there are so many variations of zinc fixatives, and none are what we need. Any help would be appreciated. Thank you! * Kathy, What you are looking for is the true, original formulation of the Beckstead Zinc Tris buffer (non-formalin) fixative. The BD Biosciences Zinc Fixative (Formalin Free) Cat# 552658 technical data sheet cites his publication, but the MSDS shows zinc acetate is not part of the reported toxic substances. If they are citing Beckstead, they are more than likely using his original formulation (see below) but to make sure this is absolutely correct, you should contact their technical services about this. They have zinc in the recipe from zinc chloride and maybe they have modified the formulation so that it works as well or better from the original without the zinc acetate. Making it up in house was not difficult when we tried the fixative. Zinc Fixative (JB Fixative or ZSF) 0.1M Tris Buffer, pH 7.4 Tris Base 12.1 g (TRIZMA) 1N HCL --- 81.5 ml Distilled water -- 900 ml Mix to dissolve. Adjust pH to 7.4 Zinc Fixative Calcium Acetate -- 0.5 g Zinc Acetate -- 5.0 g Zinc Chloride -- 5.0 g 0.1M Tris Buffer made above -- 1000 ml Mix to dissolve. The final pH will be approximately 6.5-7.0. Do not readjust the pH, as this will cause the zinc to come out of solution. Store Zinc Fixative at room temperature. Fix tissues for 24 to 48 hours. Fixation longer than 48 hours may make the tissue brittle and difficult to cut. As far as I know, there is no other company in the US that makes up this fixative - a unique one of a kind fixative not commonly used by many labs except maybe research facililites. I think it is available from sources in Europe, but can't be sure of exactly what they are selling from publications I have read. Sorting that out was not fun. Personally, I would trust the BD Bioscience Zinc fixative (formalin free) simply because they do cite Beckstead's publication. I know that Ray Koelling, now at Phenopath, has used this fixative in the past, and he may have purchased it from BD. Hopefully he is looking in and can address your problem. He has been CC'd with this message. Good luck and hope your headache goes away - Gayle M. Callis ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] TUNEL_FFPE_very_long OT_Delete_if_uninterested
Hi Jerry, I would never try to persuade anyone. I'm no smarter than the next lab worker trying to make sense of this and science biology in general. But this is how I see TUNEL and FFPE and after all the years I'm happy with things. I have not noticed false positives at all when looking at TUNEL FFPE versus frozen or whole cells and even when adapting the animal model to other verifying procedures (annexinV in flow) or caspace-3 when applicable. Formalin can cause strand breaks, that is true but these are not amplifiable breaks. For TdT to work, the break must be of particular kinds. But that discussion is far beyond the realm of possibility in HistoNet. Up at UW, if you haven't done so, there is a great Cell and Molecular Biology Grad level course that is required in grad school and I'd recommend it for anyone. It is phenomenal to help in understanding molecular mechanisms. When I was down the hallway in grad school in Biological Structure, we had a PhD/MD student in adjacent lab who was working on apoptosis and macrophage scavenging in the developing limb buds (the web between fingers) for a thesis. Did TUNEL on FFPE and it was exquisite, beautiful and accepted as a (small part) of thesis. Thousands of peer-reviewed articles in prestigous journals use TUNEL on FFPE. Indeed, a review of specific articles where they are are trying to measure precisely strand breaks of DNA in UVA skin or germ-cell or other models, every one I see, the specimen is then placed into formalin or such for processing. Why would they try to get a precise measurement of DNA strand breaks in their experimental model only to throw specimens into formalin if that is going to cause ubiquitous strand breaks and flood the assay? All DNA strand breaks are not the same and the kind caused by formalin are simply not amplifiable by TdT. If they were, you could simply place a piece of normal, healthy mouse liver or some other tissue with non-apoptosing cells in formalin, process and every nuclei in liver, with broken strands from formalin would turn positive. I've, never, ever seen such a thing in a well set up TUNEL assay. And then if you do something to cause apoptosis, those nuclei are positive while others remain unaffected by formalin. Since I worked in thymus a lot,I had a control block of 4 thymii, one no treatment and the other 3 with varying time treatments of hydrocortisone to induce steroidal T-cell apoptosis (widely accepted model). The differences where easily recognizable in apoptosis and when run with other tissues, non-apoptosing cells were still clean as anything. (1) strand breaks by formalin not amplifiable in TUNEL, otherwise every nuclei formalin fixed would be pos. Just not so. (2) microtomy could not possibly cause strandbreaks that are amplifiable. Cutting is working on a micron scale. Pieces of DNA are at nanometer or Angstrom scale. If it did, again, every nuclei would be possitive that the blade sliced through, And then why would frozen TUNEL be ok if a blade is slicing through DNA in those sections. So I disagree that every one of them (nuclei in section) is detectable by TUNEL. Again as before a common piece of healthy mouse liver cut at 4 microns would show nuclei all over positive. That just doesn't happen. What I've seen and done, with very specifically controlled experiments, the vast amount of peer-reviewed literature and thinking through the processes at a molecular level, I just can't come to the conclusion that TUNEL on FFPE is a failed assay that cannot work. No assay is perfect. PCR has primer-dimer and other problems to deal with. IHC has false pos and false neg to worry about. Flow has Fc receptor problems to deal with. ISH has stringency issues to create false pos and false neg. I think there is beyond overwheming evidence that TUNEL on FFPE is an essential (if never perfect) tool in molecular science for apoptosis but as with every assay you have to be aware of limitations and problems. I just don't believe at all that amplifiable formalin strand breaks and amplifiable microtome strand beaks are any problem at all and should not be a reason to turn from TUNEL on FFPE. But again, that is just my opinion that is no more valid than others who might differ. Ray Raymond Koelling PhenoPath Labs Seattle, WA 98155 rocedure called microtomy. When a microtome bvlade passes through the nucleus of a cell it breaks a lot of DNA strands. And every one of them is detectable by TUNEL. I've heard of people getting rerasonable results with whole cells and frozen tissues, by for FFPE tissue, my current philosophy is: It is an assay that CANNOT work, even in principle. 'Course, I've been wrong about other things, I'm open to persuasion. Jerry Ricks Research Scientist University of Washington Department of Pathology
Re: [Histonet] anyone using Promega TUNEL assay?
Tyrone, I agree with Amos Brooks about the Chemicon kit and I agree with Jason Palmer about about Promega, especially in regards to smaller, controlled volumes to decease cost per slide. In addition, I only used both those kits directions as starting points. Took care of the proteinase digestion optimized to my standards for my controls in my lab and also emperically found a lesser amount (much less than suggested) of Tdt was preferable. In fact, I could attribute false-positive staining to use of kit suggested amounts of Tdt. Backing off on that concentration eliminated false positives while retaining strong pos staining. And made kit last longer. As for working up caspace-3, I'm sure you aware of the caveat that (1) brain especially can utilize non-canonical apoptosis pathways (caspace-3 independent pathways that leads to false negatives in the assay) and (2) there is now clear and convincing evidence that caspace-3 activity in brain has been linked to other things aside from apoptosis such as synaptic plasticity modulation and other non-apoptotic but normal brain functions. Ray Koelling PhenoPath Labs Seattle, WA - Original Message - From: Tyrone Genade tgen...@gmail.com To: histonet histonet@lists.utsouthwestern.edu Sent: Saturday, July 11, 2009 6:11:53 AM GMT -08:00 US/Canada Pacific Subject: [Histonet] anyone using Promega TUNEL assay? Hello, For my PhD project my Prof and I are entertaining doing some TUNEL work on our brain sections in search of apoptotic nuclei but the kit is very expensive and would like to get some more information from some one who is using one of the kits and doesn't have a financial interest in selling us one. Locally, the Promega Kit has the highest tests to cost ratio. Anyone out there using this kit and willing to share their experience? -- Tyrone Genade http://tgenade.freeshell.org email: tgen...@freeshell.org tel: +27-84-632-1925 (c) Romans 6:23: The gift of God is eternal life through Christ Jesus our Lord. To find out how to receive this FREE gift visit http://www.alpha.org. ___ 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] direct-conjugates for IHC
Jennifer, In the past, upon occasion when need arose, I'd use directly conjugated primary antibodies. Mainly biotin or dig or peroxidase but also FITC directly to primary. Then can look at it fluorescently or come back with an anti-FITC of some kind (several are good). Used these for my flow but also a lot of IHC. Have never tried the Texas Red route for IHC. What you say is true; direct conjugation can end up giving weaker signal but it doesn't have to be so bad. 1) If you are doing the conjugation, that is great and you have a chance to play with the system. Conjugation can result in near loss of antibody to bind or can result in good Ab binding or if you have the knack it can result in little loss of binding. For flow, the conjugation is not critical since as long as you don't ruin the antibody, it will work since flow reagents are generally used in excess since there is no background (the background is plain sheath fluid) although certainly you can put in too much. For IHC you need to be gentler in conjugation. Don't know your company but if a biotech you might have a BiaCore machine (measure affinity of Ab's) or there are other ways to measure affinity. So I would always keep an aliquot of native antibody and after I did my conjugation, give those people both samples to measure affinity (or how much loss). Tell me immediately if I lost too much Ab binding ability because of my (hopefully not poor) conjugation. In general yes, directly conjugating your primary can weaken signal but it doesn't have to be as much as conventional wisdom or lore makes it. Raymond Koelling PhenoPath Labs Seattle, WA - Original Message - From: Jennifer Anderson jander...@halozyme.com To: histonet@lists.utsouthwestern.edu Sent: Monday, May 4, 2009 12:04:57 PM GMT -08:00 US/Canada Pacific Subject: [Histonet] direct-conjugates for IHC Hello everyone! I work with a scientist who insists on using primary antibodies directly conjugated with FITC or Texas Red for IHC. In my past experience these directly conjugated antibodies didn't give a strong enough signal for use in IHC (I've seen them used only for FACS analysis). I would appreciate your professional comments. Thanks again, so much, for your insight! Jennifer M. Anderson, Scientist Halozyme Therapeutics, Inc. 11404 Sorrento Valley Road San Diego, CA 92121 858-704-8333 jander...@halozyme.com mailto:jander...@halozyme.com The information transmitted in this email is confidential and is intended only for the person(s) or entity to which it is addressed. Delivery of this message to any person other than the intended recipient(s) is not intended in any way to waive confidentiality or any applicable privilege. Any review, retransmission, dissemination or other use of, or taking of any action in reliance upon, this information by individuals or entities other than the intended recipient is prohibited by Halozyme and may be in violation of applicable laws. If you received this in error, please contact the sender and delete/destroy this email. ___ 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] Lab Week-Histology Trivial or Fun Facts LONG OFF TOPIC
Hi in grad school taking microanatomy and pathology classes, 2 that I heard are this: The surface area of all the alveoli in the lungs of an adult is between 40-70 square meters. That seems reasonable in having a 40-70 square meter surface (where all gas exchange takes place) represent all the gas exchange in lungs. Have seen that figure numerous times so while can't test it, can believe it. The other one that I also can't test and is hard to believe is that the sum total length of all vessels (large small, artery vein down to every single capillary) in one adult measures about 100,000 kilometers (62,000 miles). Again there are many disparate medical and anatomical references so either all right or all wrong. The 2 micron sectioned egg I don't believe. (1) There are 25,400 microns in an inch. A 2 inch long egg is about 50,000 microns long. At 2 microns per section thats about 25,000 egg sections. Even is each section is 2 square inches (that's generous since each end isn't close to 2 squre inches in area), thats 100,000 square inches. At 1,296 square inches per square yard, that's about 40 square yards which is far short of a football field (100 yards x 53 yards). (2) If you calculate the volume of a solid rectangle covering a football feild that is 100 yards x 53 yards x 2 microns and of course converting all to yards or microns, the answer is a specific volume. If you take the volume of an ellipsoid which is four thirds times pi times a times b times c with a, b and c being the lenggth of the 3 axis of the ellipsoid, and using approximate measurements for the egg, I come up with far , far less volume in egg than in the rectangular solid covering football field. (3) This is a classical calculus definte integral washer problem. Whether this egg as an ellipsoid is scalene, oblate or prolate, integrating volume over the limits of integration gives me much, much less volume than is needed to cover a football field 2 microns thick. Have tried all 3 methods and converting everything to microns or yards using scientific notation. So 6 calculations. Everytime I come up somewhere close to the area of 2 micron slices covering approximately 1/100 of the football field. Unless my math is all wrong, or this is a humongous, enormous ostrich and not chicken egg. Ray Raymond Koelling PhenoPath Labs Seattle, WA - Original Message - From: Disher Lori lori.dis...@hcahealthcare.com To: histonet@lists.utsouthwestern.edu Sent: Tuesday, April 14, 2009 12:12:40 PM GMT -08:00 US/Canada Pacific Subject: [Histonet] Lab Week-Histology Trivial or Fun Facts Hello, I was wondering if anyone has some histo trivial-fun facts to share for Lab Week? I remember a supervisor told me long ago that she was told while in training, that if you took a hard boilded egg and sectioned it at 2 microns you would have enough sections to cover a football field. Has anyone ever heard that one before? Can anyone contribute any others? We are trying to come up with some games for lab week. Lori ___ 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] FW: CD86 - mouse
Joost, I was curious myself what was out there and maybe someone has answered you but I haven't seen anything on the HistoNet. Haven't done anything with murine CD86 (B7.2) for 3-4 years but this is what I can recall. Of course you know the whole CD80/86/CD28/CTLA-4 costimulatory story is tough because of transient nature and upregulations during stimulation or disease. Using the GL-1 clone from BD (don't work for them but like their anti-mouse reagents), we got pretty good results on frozen mouse tissues with standard frozen fixation and IHC. At least reasonable in that staining corresponded to flow data from similar mouse tissues. As far as FFPE mouse tissues, the best I could do was to get pretty good but not overly convincing data with retieval but with a tyramide amplification necessary. In flow if you look at a one color diagram of staining (isotype and then GL-1) the peaks are not well separated even with something like LPS stimmed cells. In fact, sometimes the pos peak might move a log but the base of the peaks (isotype and GL-1) can overlap substantially. On a flow diagram, shoulders and mean peak heights are easy to differentiate over neg or background but I could never really seperate out truely pos from truely negative from low positive in FFPE mouse like you can with flow. So frozen was the way we went. And I know there is, at least in humans, soluble CD86 floating around due to shedding/alternative splicing. If that happens in mice (I'm clueless) that could contribute to background. GL-1 clone in frozen mouse tissue was how I ended up going but haven't touched this for many years. Ray Raymond Koelling PhenoPath Labs Seattle, WA - Original Message - From: J.P. Bruijntjes (Joost) joost.bruijnt...@tno.nl To: histonet@lists.utsouthwestern.edu Sent: Wednesday, March 18, 2009 7:48:00 AM GMT -08:00 US/Canada Pacific Subject: [Histonet] FW: CD86 - mouse Hi histonetters Is really nobody working with cd86 on mouse tissues? Joost Bruijntjes TNO Quality of Life Zeist Holland TNO.NL http://www.tno.nl/ Joost Bruijntjes T +31 30 694 44 80 F +31 30 694 49 86 E joost.bruijnt...@tno.nl mailto:joost.bruijnt...@tno.nl Disclaimer http://www.tno.nl/tno/email/ This e-mail and its contents are subject to the DISCLAIMER at http://www.tno.nl/disclaimer/email.html ___ 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] expression pattern of GFP fusion protein in cells and mouse
DONGTAO FU, Was hoping to see some response for curiosity sake but haven't so this is my take. In a former life have seen such an occurence. Not with these same reagents but similar happenings. And while I didn't take the time to investigate exactly why, we attributed it to posttranslational modifications. Those 293 cells are human kidney cells and while cells they are certainly different from mouse cells. When you put in that AAV/dystrophin/GFP cassette into 293 or mice or any kind of other expression model, while they might be transcribed similarly, each cell type (species) has it's own unique way of posttranslational modifications to the protein. Acetylation, alkylation, glycosylation, and hundreds of others, any one of which could modify an epitope or cover it up. There are articles innumerable on how posttranslational modifications, different in different species, can affect protein composition and shape (and obviously that could very well impact ability to see it with an antibody that might work perfectly well in detection in another system). Ray Koelling PhenoPtah Labs Seattle, WA - Original Message - From: DONGTAO FU f...@ufl.edu To: histonet@lists.utsouthwestern.edu Sent: Thursday, February 19, 2009 6:48:58 AM GMT -08:00 US/Canada Pacific Subject: [Histonet] expression pattern of GFP fusion protein in cells and mouse Hi, all A researcher met a problem of GFP fusion protein expression and is searching an answer from the specialists here. The following is his question: I am working with an AAV vector which contains a transgene (dystrophin) which is fused to GFP. When I inject this vector in mice, and stain the tissues with antibodies raised against GFP and dystrophin, I only pick up GFP. It is blazing. I was surprised not to see colocalization given that this is a fusion protein. (I used the same immunostaining protocol that others have successfully used before me to pick up dystrophin). So, the next thing I did was infect 293 cells with the same vector. When I stained those cells, I picked up beautiful expression of dystrophin and GFP. They were perfectly colocalized, as expected. So, my question is, have you seen examples where certain epitopes are masked in one cellular environment and not in another? (maybe the reason that I cannot detect the dystrophin in the mouse tissues, but I can in 293 cells). Does anyone know the possible answer of this phenomenon? Any thoughts will be greatly appreciated. Ann ___ 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] Klotskin's modification of the Masson's Trichrome
Isn't a Klatskin tumor (of the biliary tree) spelled with A and not O? There are multiple pictures and references to Klatskin's Trichrome. Try A spelling and not O. Ray Koelling Research Pathology PhenoPath Labs Seattle -- Original message -- From: Pat Laurie foreig...@gmail.com Hi Everyone, Has anyone heard of the Klotskin's modification of the Masson's Trichrome? I am working with a liver pathologist who wants me to try to work it up. I can't seem to find it anywhere. I have tried numerous books, and scoured the internet. Someone asked about 3 years ago on histonet, but there were no replies. If anyone could help me I would appreciate it. Thanks, -- Patrick Laurie (HT, ASCP) Cellnetix Pathology and Laboratories Seattle, Wa ___ 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
