RE: [Histonet] MSI on Ventana
Hey Tony, We are using VMS antibodies (2 are manufactured by CellMarque) on our Ultras. We've had to go with the Optiview detection to get satisfactory results. Let me know if you'd like our protocols. Linda A. Sebree University of Wisconsin Hospital Clinics IHC/ISH Laboratory 600 Highland Ave. Madison, WI 53792 (608)265-6596 FAX: (608)262-7174 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tony Reilly Sent: Monday, July 08, 2013 9:47 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] MSI on Ventana Hello We have previously been sending our MSI Abs to another lab where they are being performed on the Bond. We have now been informed that we are to do our own and one of my IHC staff with previous experience in another lab informs me that they do not work as well on the Ventana platforms that we are currently running. Is ther anybody out there successfully staining MSI on the Ventana. If so what brand/clone of Abs are you using? We are trying to avoid using the Ventana RTU Abs due to cost. regards Tony Tony Reilly B.App.Sc. , M.Sc. Chief Scientist, Anatomical Pathology Pathology Queensland-PA Laboratory Health Services Support Agency | Department of Health Level 1, Building 15,Princess Alexandra Hospital Ipswich Road,WOOLLOONGABBA Qld4102 Ph: 07 3176 2412 Mob: 0402 139411 Fax: 07 3176 2930 Email: tony_rei...@health.qld.gov.au Web: www.health.qld.gov.au/qhcss/ This email, including any attachments sent with it, is confidential and for the sole use of the intended recipient(s). This confidentiality is not waived or lost, if you receive it and you are not the intended recipient(s), or if it is transmitted/received in error. Any unauthorised use, alteration, disclosure, distribution or review of this email is strictly prohibited. The information contained in this email, including any attachment sent with it, may be subject to a statutory duty of confidentiality if it relates to health service matters. If you are not the intended recipient(s), or if you have received this email in error, you are asked to immediately notify the sender by telephone collect on Australia +61 1800 198 175 or by return email. You should also delete this email, and any copies, from your computer system network and destroy any hard copies produced. If not an intended recipient of this email, you must not copy, distribute or take any action(s) that relies on it; any form of disclosure, modification, distribution and/or publication of this email is also prohibited. Although Queensland Health takes all reasonable steps to ensure this email does not contain malicious software, Queensland Health does not accept responsibility for the consequences if any person's computer inadvertently suffers any disruption to services, loss of information, harm or is infected with a virus, other malicious computer programme or code that may occur as a consequence of receiving this email. Unless stated otherwise, this email represents only the views of the sender and not the views of the Queensland Government. ** ___ 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] Paraplast vs. Paraplast plus
Does anyone have a preference over Paraplast and Paraplast plus for mouse tissues? Thanks!! Mike ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Plastics- please help!
Hello histonetters, Our lab is thinking about introducing plastics but we are not sure what we need or how to set it up. Basically everything on how to set up a plastics lab. I don't know anything about it but would love to learn. Can someone please help us! ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Plastic/Plexiglass Incubation chamber for IHC slides
Dear All: We use a Tupperware container, with a nice air/liquid-tight lid. But any container like that will work. Our trick is to buy plastic ceiling tile material from the hardware store. (Used in ceiling panels.) It comes in big sheets (5 feet by 3 feet?) and is pretty cheap (~$12/sheet). We get the white or clear kind that has square holes cut through the plastic. The holes are about 1 cm square. We then use pliers to cut out panels/pieces that will fit inside the container (wear face protection because the plastic flies all over the place). We put one piece in the bottom of the container, then set a metal slide rack (or any water-proof slide rack) on top. We put water in the bottom and this first panel keeps the slides from getting wet. Then, I cut out 4 single squares of the material and put them inside the metal slide racks, at all 4 corners. Then I can lay another metal slide rack on top. Another 4 pieces, another rack, etc. So you can build up a tall stack depending on the size of your container. And the ceiling tile material is also really good for under drying dishes and other uses around the lab. The cheap way to make an incubation chamber! Sarah Pixley Univ. of Cincinnati ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Validating a new reagent
My lab is considering getting our xylene and formalin from a different company so all of our reagents are purchase through only one company. I know we cannot just start using these new reagents without doing some kind of validation with our processor and stainer, but after thorough searching on CAP I cannot for the life of me find what we need to do to start using these reagents. Can someone give me some direction as to where to find this info, or if anyone knows what to do let me know? Thanks, Brittany Reetz, HT (ASCP)cm Diagnostic and Treatment Center 3401 Cranberry Blvd Weston WI 54476 (715)-393-2081 reet...@dxandtx.commailto:reet...@dxandtx.com CONFIDENTIALITY NOTICE: This e-mail communication and any attachments may contain confidential and privileged information for the use of the designated recipient(s) named above. If you are not the intended recipient, you are hereby notified that you have received this communication in error and that any review, disclosure, dissemination, distribution or copying of it or its contents is prohibited. If you have received this communication in error, please notify the sender at the electronic mail address noted above and destroy all copies of this communication and any attachments. Thank you for your cooperation. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Plastic/Plexiglass Incubation chamber for IHC slides
Sarah, Wow, brings back memories of the good ol' days when we used all kinds of stuff to make our own trays. In one lab I worked at we had a small Tupperware container for each antibody and layed paper towels and wood rods on the bottom to hold the slides the surface. We'd wet the paper towels for humidity. When a new lab was built they specifically designed it for this method and we had a 20-foot long bench just so we could line up dozens of these small Tupperware containers! What a pain that was to go through each round of washing!! We did 150 slides a day that way for years. Blessed is the day we got automated stainers!! Tim Morken Supervisor, Electron Microscopy/Neuromuscular Special Studies Department of Pathology UC San Francisco Medical Center -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Pixley, Sarah (pixleysk) Sent: Tuesday, July 09, 2013 10:24 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] RE: Plastic/Plexiglass Incubation chamber for IHC slides Dear All: We use a Tupperware container, with a nice air/liquid-tight lid. But any container like that will work. Our trick is to buy plastic ceiling tile material from the hardware store. (Used in ceiling panels.) It comes in big sheets (5 feet by 3 feet?) and is pretty cheap (~$12/sheet). We get the white or clear kind that has square holes cut through the plastic. The holes are about 1 cm square. We then use pliers to cut out panels/pieces that will fit inside the container (wear face protection because the plastic flies all over the place). We put one piece in the bottom of the container, then set a metal slide rack (or any water-proof slide rack) on top. We put water in the bottom and this first panel keeps the slides from getting wet. Then, I cut out 4 single squares of the material and put them inside the metal slide racks, at all 4 corners. Then I can lay another metal slide rack on top. Another 4 pieces, another rack, etc. So you can build up a tall stack depending on the size of your container. And the ceiling tile material is also really good for under drying dishes and other uses around the lab. The cheap way to make an incubation chamber! Sarah Pixley Univ. of Cincinnati ___ 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] p16 control tissue
We are going to be running the p16 antibody from Biocare (RUO) on skin specimens. What type of tissue should I run for a positive control? The data sheet says normal testis.. Laurie Colbert ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] immuno moisture chambers
Ted Pella has some options. We use the Immunostain Moisture Chambers #21049, but there are others. http://www.tedpella.com/glasswar_html/slidedsh.htm#21049 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: p16 control tissue
We are using Biocare's ab with squamous cell ca as the control of choice. I believe you can also use cervical dysplasia or tonsil. Tom McNemar, HT(ASCP) Histology Co-ordinator Licking Memorial Health Systems (740) 348-4163 (740) 348-4166 tmcne...@lmhealth.org www.LMHealth.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie Colbert Sent: Tuesday, July 09, 2013 1:50 PM To: Histonet Post (histonet@lists.utsouthwestern.edu) Subject: [Histonet] p16 control tissue We are going to be running the p16 antibody from Biocare (RUO) on skin specimens. What type of tissue should I run for a positive control? The data sheet says normal testis.. Laurie Colbert ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This e-mail, including attachments, is intended for the sole use of the individual and/or entity to whom it is addressed, and contains information from Licking Memorial Health Systems which is confidential or privileged. If you are not the intended recipient, nor authorized to receive for the intended recipient, be aware that any disclosure, copying, distribution or use of the contents of this e-mail and attachments is prohibited. If you have received this in error, please advise the sender by reply e-mail and delete the message immediately. You may also contact the LMH Process Improvement Center at 740-348-4641. E-mail transmissions cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] RE: Plastic/Plexiglass Incubation chamber for IHC slides
We use bioassay dishes with 10mL pipets glued to them. The slides sit on two pipets and you can get two rows of slides in. Cutting the pipets' ends off is the hardest part. I take that back, finding a glue that will hold the pipets has been a problem lately, as rubber cement isn't made like they used to make it. A glue gun works, but sometimes the pipets need to be reglued. Emily By bitching and bitching and bitching, they could exhaust the drama of their own horror stories. Grow bored. Only then could they accept a new story for their lives. Move forward. -Chuck Palahniuk, Haunted On Tue, Jul 9, 2013 at 1:34 PM, Morken, Timothy timothy.mor...@ucsfmedctr.org wrote: Sarah, Wow, brings back memories of the good ol' days when we used all kinds of stuff to make our own trays. In one lab I worked at we had a small Tupperware container for each antibody and layed paper towels and wood rods on the bottom to hold the slides the surface. We'd wet the paper towels for humidity. When a new lab was built they specifically designed it for this method and we had a 20-foot long bench just so we could line up dozens of these small Tupperware containers! What a pain that was to go through each round of washing!! We did 150 slides a day that way for years. Blessed is the day we got automated stainers!! Tim Morken Supervisor, Electron Microscopy/Neuromuscular Special Studies Department of Pathology UC San Francisco Medical Center -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto: histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Pixley, Sarah (pixleysk) Sent: Tuesday, July 09, 2013 10:24 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] RE: Plastic/Plexiglass Incubation chamber for IHC slides Dear All: We use a Tupperware container, with a nice air/liquid-tight lid. But any container like that will work. Our trick is to buy plastic ceiling tile material from the hardware store. (Used in ceiling panels.) It comes in big sheets (5 feet by 3 feet?) and is pretty cheap (~$12/sheet). We get the white or clear kind that has square holes cut through the plastic. The holes are about 1 cm square. We then use pliers to cut out panels/pieces that will fit inside the container (wear face protection because the plastic flies all over the place). We put one piece in the bottom of the container, then set a metal slide rack (or any water-proof slide rack) on top. We put water in the bottom and this first panel keeps the slides from getting wet. Then, I cut out 4 single squares of the material and put them inside the metal slide racks, at all 4 corners. Then I can lay another metal slide rack on top. Another 4 pieces, another rack, etc. So you can build up a tall stack depending on the size of your container. And the ceiling tile material is also really good for under drying dishes and other uses around the lab. The cheap way to make an incubation chamber! Sarah Pixley Univ. of Cincinnati ___ 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: p16 control tissue
We use a nice CIN3 on a cervical cone. Daniel Hewitt Histology Supervisor, HVS 412-749-7371 This email, 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, or an agent responsible for delivering the message to the intended recipient, please contact the sender by reply e-mail and delete and destroy all copies of the original message, including attachments. Please note that any views or opinions presented in this e-mail are solely those of the author and do not necessarily represent those of Heritage Valley Health System. The integrity and security of this message cannot be guaranteed on the internet. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tom McNemar Sent: Tuesday, July 09, 2013 1:59 PM To: 'Laurie Colbert'; Histonet Post(histonet@lists.utsouthwestern.edu) Subject: [Histonet] RE: p16 control tissue We are using Biocare's ab with squamous cell ca as the control of choice. I believe you can also use cervical dysplasia or tonsil. Tom McNemar, HT(ASCP) Histology Co-ordinator Licking Memorial Health Systems (740) 348-4163 (740) 348-4166 tmcne...@lmhealth.org www.LMHealth.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie Colbert Sent: Tuesday, July 09, 2013 1:50 PM To: Histonet Post (histonet@lists.utsouthwestern.edu) Subject: [Histonet] p16 control tissue We are going to be running the p16 antibody from Biocare (RUO) on skin specimens. What type of tissue should I run for a positive control? The data sheet says normal testis.. Laurie Colbert ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This e-mail, including attachments, is intended for the sole use of the individual and/or entity to whom it is addressed, and contains information from Licking Memorial Health Systems which is confidential or privileged. If you are not the intended recipient, nor authorized to receive for the intended recipient, be aware that any disclosure, copying, distribution or use of the contents of this e-mail and attachments is prohibited. If you have received this in error, please advise the sender by reply e-mail and delete the message immediately. You may also contact the LMH Process Improvement Center at 740-348-4641. E-mail transmissions cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. Thank you. ___ 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] TEMP POSITION PALM BEACH, FL
Hi...We're looking for a Histotechnologist for a 3-4 month temporary (possible full-time) position at our very busy dermatology lab in Delray Beach, FL. THIS IS NOT AN AGENCY. Please contact me below or send your resume if interested. This would be a 8a-5p, full time position from July-November. Here is our current advertisement. Job Duties Include: Grossing, processing, embedding, cutting, staining and coverslipping. Accessioning all specimens along with other team members. Attention to detail. Performing special stains, following protocols. Putting slides together with requisitions- double checking for accuracy. Cutting control slides Quality Control Adhering to all Policy and Procedures Job Requirements Requirements Include: Current Florida Histotechnologist license A MINIMUM of Five years + experience in the following: Embedding Cutting Staining Special Stains Troubleshooting Immunohistochemistry- 2 years + experience preferred. Grossing experience preferred, but will train. PLEASE DO NOT CONTACT ME FROM ANY AGENCIES, YOU WILL NOT BE ANSWERED. Thanks! Kari M Simeone Histology/Immunohistochemistry Specialist Supervisor Alternate Laboratory Supervisor ADCS Clinics, LLC Advanced Dermatology and Cosmetic Surgery Ameriderm 561.819.0857 ext 100 561.819.6517 fax ksime...@leavittmgt.com www.advancedderm.com http://www.advancedderm.com/ The information contained in this message and any attachments is intended only for the use of the individual or entity to which it is addressed, and may contain information that is privileged, confidential and exempt from disclosure under applicable law. If you have received this message in error, you are prohibited from copying, distributing or using the information. Please contact the sender immediately by return e-mail and delete the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] COX/SDH Combo staining
Hi I'm looking for COX/SDH combo staining procedures for muscle enzymes we currently do these two enzyme procedure separately and we our looking into combining them or any idea as to what sites I may find the information. Thanks Helene deg...@upstate.edu ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Plastic/Plexiglass Incubation chamber for IHC slides
For Manual IHC I suggest best the Sequenza Slide Rack from Thermo Scientific. It makes your life easier and you can save money, because you only need 100 ul of any reactive per slide. Here is the link to the web page. http://www.thermo.fi/com/cda/product/detail/0,1055,105630785,00.html I hope it helps. Cesar Romero Buenos Aires Argentina ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC controls
So, I am working on some IHC stuff. I need control for mouse tissues. While I have just about every normal tissue you could ask for, several of these antibodies say the positive control is lung cancer, or breast cancer, or prostate cancer. While I have all of these in human I don't have them in the species I am staining in...mouse. Also, a couple of them say tonsil...do mice even have tonsils?? Anyhoo, what is ya'lls suggestion on this one? Do I stain the appropriate tissue just in the wrong species (human), or does someone know where I can purchase mouse control slides and/or blocks? Thanks Sarah Goebel-Dysart, BA, HT(ASCP), QIHC (ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Section cells on a plastic trans well membrane
Hi All, Has anyone tried to section cells cut from a trans well dish? We have cells that are cultured and attach to a trans well membrane and our pathologist wants to look at them on the membrane but the membrane is a thin plastic film...see the problem. We fixed the cells in the trans well for 5 minutes then cut the trans well out of the well and bisected it and processed overnight we use paraffin type #1 paraffin. The next morning we embedded it with type #9 paraffin. The samples cut but do not stay on the slide after xylene. I think the plastic does not adhere to the slide. Would a adhesive slide work to keep the thin plastic stuck to the slide?? My pathologist does not want to change to a collagen membrane so he wants me to find another (harder paraffin) to use, I don't think that will help. Has anyone does similar processing/embedding? I'll take any help I can get.. Thanks, Jamie Abbvie Bioreseach Center Pharmacology 100 Research Dr. Worcester,Ma 01605 OFFICE+1 508-688-3134 FAX +1 508-793-4895 EMAIL jamie.erick...@abbvie.commailto:jamie.erick...@abbvie.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Section cells on a plastic trans well membrane
Jamie We have sectioned these before and we used charged slides and did not have a problem with section adherence. I would fix longer than 5 minutes. We would use biopsy punches to remove the membrane from the well we would process the disc whole between biopsy pads and bisect prior to embedding. You need to use a good plus slide and it you are still having problems I would let the unstained slides sit for a couple days prior to staining. Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 881-0763 cell (303) 682-9060 fax l...@premierlab.com Ship to address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Erickson, Jamie E [jamie.erick...@abbvie.com] Sent: Tuesday, July 09, 2013 2:52 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Section cells on a plastic trans well membrane Hi All, Has anyone tried to section cells cut from a trans well dish? We have cells that are cultured and attach to a trans well membrane and our pathologist wants to look at them on the membrane but the membrane is a thin plastic film...see the problem. We fixed the cells in the trans well for 5 minutes then cut the trans well out of the well and bisected it and processed overnight we use paraffin type #1 paraffin. The next morning we embedded it with type #9 paraffin. The samples cut but do not stay on the slide after xylene. I think the plastic does not adhere to the slide. Would a adhesive slide work to keep the thin plastic stuck to the slide?? My pathologist does not want to change to a collagen membrane so he wants me to find another (harder paraffin) to use, I don't think that will help. Has anyone does similar processing/embedding? I'll take any help I can get.. Thanks, Jamie Abbvie Bioreseach Center Pharmacology 100 Research Dr. Worcester,Ma 01605 OFFICE+1 508-688-3134 FAX +1 508-793-4895 EMAIL jamie.erick...@abbvie.commailto:jamie.erick...@abbvie.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] Wage and Vacancy Survey
I was able to locate the vacancy survey that the ASCP published, but cannot find the wage survey. Does anyone have information? It was supposed to be published in the November 2012 issue of LabMedicine. Thanks, Jennifer MacDonald ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] COX/SDH Combo staining
Here is our technique: Cytochrome Oxidase - SDH Use 1. Defects of cytochrome oxidase activity 2. Demonstration of mitochondria Underlying Principle Combining the COX with the SDH can demonstrate COX negative fibres. These may be SDH positive and may be ragged red fibres. 3 of the 13 subunits of COX are encoded by mitochondrial DNA whereas SDH is encoded by nuclear DNA. Therefore SDH is not affected by mitochondrial DNA mutations. Ragged red fibres in mitochondrial myopathies are generally COX neg (except in MELAS). Intra fibre mosaicism (mixture of bluish COX deficient and brownish COX positive mitochondria within the same fibre are also well demonstrated by this combined technique). Fixation and Sectioning Air dried unfixed 8µM cryostat sections Reagents 1. PBS Buffer: Dissolve one Dulbecco PBS Tablet in 100ml distilled water 2. COX A Solution: Warning: DAB is carcinogenic – Avoid contact with skin Sucrose 0.75g 3,3' Diaminobenzidine.4HCl (DAB)0.05g Adjust to pH 7.6 PBS Buffer up to 50ml Aliquot in 2ml amounts (labelled CA), store at -20oC 3. COX B Solution: Catalase (Sigma C9322)0.001g Cytochrome C (Sigma C2037)0.05g Adjust to pH 7.6 PBS Bufferup to 50ml Aliquot in 2ml amounts (labelled CB), store at -20oC 4. Incubating medium Defrost one CA and one CB vial, mix and place in a 2-slide plastic slide mailer. Place mailer in a coplin jar for support. 5. SDH Reagents 1 Stock 0.2M Sodium Dihydrogen Phosphate (NaH2PO4) Sodium Dihydrogen Phosphate 23.996 g Distilled water 1000 ml Store at room temperature 2 Stock 0.2M Disodium Hydrogen Phosphate (Na2HPO4) Disodium Hydrogen Phosphate 28.392 g Distilled water 1000 ml Store at room temperature 3 0.6M succinate solution (adjusted to pH 7.6) Warning: Irritant – see MSDS Sodium succinate12.96 g Distilled water 64.00 ml 1M HCl 0.40 ml pH to 7.6 with 0.2M Disodium Hydrogen Phosphate Distilled water up to 80 ml Aliquot 800μl into eppendorf vials (labelled S) and store at -20C 3. Yellow SDH Incubation Medium Stock 0.2M Sodium Hydrogen Phosphate52ml Stock 0.2M Disodium Hydrogen Phosphate 348ml Nitro Blue Tetrazolium (NBT)0.6 g Adjust to pH 7.6 with stock phosphate solutions Aliquot 4ml into tubes labelled “SY” and store at -20C (enough for 100 tubes) 4. Incubating medium (prepare fresh) 1. Defrost a vial of succinate solution (S) and a Yellow Incubation Solution (SY) 2. Add solution “S” to “SY”, prior to use, mix and place in a small plastic slide mailer. Method 1. Prepare incubation solution 2. Immediately place frozen sectioned slides in incubation solution and incubate for two hours at room temperature. 3. Check staining and replace for longer if required 4. Rinse slides in distilled water and prepare SDH incubation medium. 5. Add slides to SDH medium and incubate at 37oC for 1-2 hour. 6. Rinse slides in distilled water. 7. Rinse in water, dehydrate, clear and mount. Results Cytochrome Oxidase positive mitochondriaBrown. Cytochrome Oxidase negative mitochondriaBlue References 1. Seligman etal (1968) J Cell Biol 38:1-14. 2. Loughlin M. (1993). Muscle biopsy. A laboratory investigation. Butterworth-Heinemann p.38-39. 3. Sheehan D, Hrapchak B. (1987). Histotechnology, 2nd Ed. Batelle Press, Columbus p306-307 Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Helene Degan Sent: Wednesday, 10 July 2013 5:33 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] COX/SDH Combo staining Hi I'm looking for COX/SDH combo staining procedures for muscle enzymes we currently do these two enzyme procedure separately and we our looking into combining
RE: [Histonet] COX/SDH Combo staining
Easier way to do this stain...since you do these two stains separately incubate the slide in a the COX solution for 1 hour rinse in distilled water and incubate in SDH solution for 1 hr. rinse 5min in physiological saline, 10min in 10% formalin saline, rinse in 15% ethanol then mount with Crystal mount media. Let dry then cover slip as usual Florence Leomiti HT (ASCP) Neuromuscular Lab Tech. Phone 503-494-6781 Fax 503-418-4249 Pager 16822 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tony Henwood (SCHN) Sent: Tuesday, July 09, 2013 4:18 PM To: 'Helene Degan'; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] COX/SDH Combo staining Here is our technique: Cytochrome Oxidase - SDH Use 1. Defects of cytochrome oxidase activity 2. Demonstration of mitochondria Underlying Principle Combining the COX with the SDH can demonstrate COX negative fibres. These may be SDH positive and may be ragged red fibres. 3 of the 13 subunits of COX are encoded by mitochondrial DNA whereas SDH is encoded by nuclear DNA. Therefore SDH is not affected by mitochondrial DNA mutations. Ragged red fibres in mitochondrial myopathies are generally COX neg (except in MELAS). Intra fibre mosaicism (mixture of bluish COX deficient and brownish COX positive mitochondria within the same fibre are also well demonstrated by this combined technique). Fixation and Sectioning Air dried unfixed 8µM cryostat sections Reagents 1. PBS Buffer: Dissolve one Dulbecco PBS Tablet in 100ml distilled water 2. COX A Solution: Warning: DAB is carcinogenic – Avoid contact with skin Sucrose 0.75g 3,3' Diaminobenzidine.4HCl (DAB)0.05g Adjust to pH 7.6 PBS Buffer up to 50ml Aliquot in 2ml amounts (labelled CA), store at -20oC 3. COX B Solution: Catalase (Sigma C9322)0.001g Cytochrome C (Sigma C2037)0.05g Adjust to pH 7.6 PBS Bufferup to 50ml Aliquot in 2ml amounts (labelled CB), store at -20oC 4. Incubating medium Defrost one CA and one CB vial, mix and place in a 2-slide plastic slide mailer. Place mailer in a coplin jar for support. 5. SDH Reagents 1 Stock 0.2M Sodium Dihydrogen Phosphate (NaH2PO4) Sodium Dihydrogen Phosphate 23.996 g Distilled water 1000 ml Store at room temperature 2 Stock 0.2M Disodium Hydrogen Phosphate (Na2HPO4) Disodium Hydrogen Phosphate 28.392 g Distilled water 1000 ml Store at room temperature 3 0.6M succinate solution (adjusted to pH 7.6) Warning: Irritant – see MSDS Sodium succinate12.96 g Distilled water 64.00 ml 1M HCl 0.40 ml pH to 7.6 with 0.2M Disodium Hydrogen Phosphate Distilled water up to 80 ml Aliquot 800μl into eppendorf vials (labelled S) and store at -20°C 3. Yellow SDH Incubation Medium Stock 0.2M Sodium Hydrogen Phosphate52ml Stock 0.2M Disodium Hydrogen Phosphate 348ml Nitro Blue Tetrazolium (NBT)0.6 g Adjust to pH 7.6 with stock phosphate solutions Aliquot 4ml into tubes labelled “SY” and store at -20°C (enough for 100 tubes) 4. Incubating medium (prepare fresh) 1. Defrost a vial of succinate solution (S) and a Yellow Incubation Solution (SY) 2. Add solution “S” to “SY”, prior to use, mix and place in a small plastic slide mailer. Method 1. Prepare incubation solution 2. Immediately place frozen sectioned slides in incubation solution and incubate for two hours at room temperature. 3. Check staining and replace for longer if required 4. Rinse slides in distilled water and prepare SDH incubation medium. 5. Add slides to SDH medium and incubate at 37oC for 1-2 hour. 6. Rinse slides in distilled water. 7. Rinse in water, dehydrate, clear and mount. Results Cytochrome Oxidase positive mitochondriaBrown. Cytochrome Oxidase negative mitochondriaBlue References 1. Seligman etal (1968) J Cell Biol 38:1-14. 2. Loughlin M. (1993). Muscle biopsy. A laboratory investigation. Butterworth-Heinemann p.38-39. 3. Sheehan D, Hrapchak B. (1987). Histotechnology, 2nd Ed.
RE: [Histonet] Wage and Vacancy Survey
I guess every other year. This is 2010 http://labmed.ascpjournals.org/content/42/3/141.full.pdf+html The last couple of years vacancy wage from ASCP site http://www.ascp.org/functional-nav/career-center Joelle Weaver MAOM, HTL (ASCP) QIHC To: histonet@lists.utsouthwestern.edu From: jmacdon...@mtsac.edu Date: Tue, 9 Jul 2013 15:42:52 -0700 Subject: [Histonet] Wage and Vacancy Survey I was able to locate the vacancy survey that the ASCP published, but cannot find the wage survey. Does anyone have information? It was supposed to be published in the November 2012 issue of LabMedicine. Thanks, Jennifer MacDonald ___ 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
