Re: [Histonet] ISH preparation
That may be what you have to do, as the first day of ISH needs to be RNase free! When I do them, it is 10 slides at a time and we have dishes and a lab bench set aside for RNase free procedures only. Obviously this is not feasible in all workplaces! I would just think about it logically. Use gloves when you can, always. Can you keep the hybridizing part RNase free? I don't know what machines you use for this--we do process the slides manually in glass dishes and then hybridize in a slide container and oven kept for RNase free purposes. After hybridization, you don't really need to be too careful. Now I'm wondering if there's some sort of magical machine that does ISH! 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 Mon, Jun 23, 2014 at 2:06 PM, jennifer.john...@genzyme.com wrote: Hi Folks! I was hoping to get a little input about preparing samples for ISH. We currently RNAse-away our tools at the time of collection, toss the tissues into formalin, process normally and then cut them using RNAse away cleaned microtomes/forceps on a bath of DEPC treated water (in a cleaned water bath). How do other labs prepare paraffin embedded samples? Do you use special reagents - formalin? Do you clean the processor and use treated reagents there too? Do you wash the slides in DEPC treated water? I am trying to figure out how far we need to go here. We are a high throughput lab and we deal with all kinds of tissues and I don't want to have to take down a processor and keep it RNAse free if I don't have to! I will run the experiments and test different methods, but I was hoping some of you could share your wisdom and give me a place to start. Thanks, JJ Jennifer Johnson Staff Scientist Genzyme Corp. Department of Pathology 5 Mountain Road Framingham, MA 01701-9322 Phn - 508-271-3610 Fax - 508-872-9080 ___ 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] BRDu IHC protocol
Hi Luis I attach my protocol for BRDu Best regards BRDU protocol 1. De-wax and hydrate through the changes of Xylenes and graded Ethanols (100%, 95%, 70%) the paraffin slides till DI water. 2. HIER :Performed in Biocare’s Decloaking Chamber with manufacture settings ( set point 1-125˚C, 30 seconds, set point 2- 90˚C , 10 seconds) in plastic container with 1X Rodent Decloaker solution which is stable modified Citrate Buffer pH 6.00, (Biocare # RD913). Optional: Place the slides in preheated till 90- 100 ˚C in 1X Rodent Decloaker solution which is stable modified Citrate Buffer pH 6.00, (Biocare # RD913) and steam 45 minutes in steamer .Remove the slides from steamer and allow them to cool till RT˚ 3. Rinse in DI water and Wash in Tris buffer three times x 5 minutes 4. Circle the tissue on the slides with hydrophobic barrier using PAP pen 5. Incubate with pepsin enzyme digestion solution – 2 minutes. 6. Prepare fresh before using: 0.25% solution of pepsin (250 mg of powder in 100 ml of Tris) (powder, Biomedicals, # 195367 ) in 1X Tris buffer( 10X Fisher Scientific, BP2471), titrated to pH 2.00 with concentrated hydrochloric acid. Optional: Preparation of 0.05M Tris(which correspond to 1X Tris) from 1M Tris (Teknova. ,cat.# T1068,pH6.8)- 10ml of 1 M Tris + 190 ml of DI water. (53 mg of powdered pepsin in 22.200 ml of Tris, pH2.5) 7. Neutralize the slides 2 times x 5 minutes each in the 0,1 M Borate Buffer , pH 8.5 (0.5M sodium Borate Buffer, Boston Bioproducts, # BB-66, 1 part of 0.5M buffer + 4 parts of DI water). 8. Wash in Tris buffer three times x 5 minutes. 9. Quench the endogenous peroxidase with PEROXIDAZED 1 (Biocare # PX 968) for 15 minutes at RT 10. Rinse in DI water and Wash in Tris buffer two times x 5 minutes 11. Block endogenous Protein with Background Sniper for 30 minutes at RT ( Biocare , #BS966) 12. Tap off the excess of protein block , do not rinse 13. Incubate the slides with the primary antibody (mouse monoclonal anti- bromodeoxyuridine, Roche, # 11 170 376001) 80 minutes. Diluent: ready-to-use antibody diluent, (Dako, # S0809). Dilutions: 1:700 Wash in Tris buffer three times x 5 minutes 14. Incubate the slides with mouse –on-mouse HRP Polymer (Biocare, #MM620) for 30 minutes at RT.-for mouse tissues or with mouse on rat HRP polymer (Biocare, # MRT 621) for rat tissues. 15. Wash in Tris buffer two times x 5 minutes 16. Stain the slides with freshly prepared DAB chromogen (Dako, #K3468) and developed end-colored product in the targeted cells under microscope. Staining is complete when the protein positive areas turn to dark brown and protein negative areas (background) remain colorless. Stop the chromogen reaction by placing the slides in DI water with following rinse in fresh DI water to remove the residual DAB and clear the slides.- 5 minutes. Galina Deyneko Novartis, Cambridge, MA 617-871-7613 w___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] New Field Based Histology Opportunities
Good Afternoon! We have had a number of new field based histology openings throughout the US. We are targeting Histology professionals who are looking for an exciting career outside of the laboratory and in the field. The company is a world leader in histology and offers a strong career track. We currently have openings in: NYC/NJ - IHC NC/SC/GA - Core Histology Midwest - Based near a major airport - Core Histology Northern California - Core Histology If you or someone you may know is interested, please send us an e-mail with your updated resume and availability to catch up and talk in more detail. Please send e-mails to m...@personifysearch.com mailto:m...@personifysearch.com . Thanks! Matt Matt Ward Program Manager Personify 5020 Weston Parkway Suite 315 Cary NC 27513 (Tel) 919.459.3654 (Tel) 800.875.6188 direct ext 103 (Fax) 919.882.8727 http://www.personifysearch.com/ www.personifysearch.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Template Special Stain validation Form or Sheet
Any suggestions or examples (from CAP, ASCP or NSH) on a template validation sheet that we could use for documentation and for CAP inspection purposes? Is there a recommended test or sample size. We have positive controls for all our test menu stains. Need a reference for all above. Ian Bernard ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] looking for tips on cutting frozen fly heads
I have another of those unusual projects and I'm looking for some advice of handling the drosophila flies before freezing (to fix or not to fix) and then freezing tips. Anybody have any experience with these flies? They brought me some this morning but the results were not good. They killed the flies in 95% ETOH and strung them on a fly collar and put them in OCT and froze with a dry ice-alcohol slurry. Looks like ice crystals and a lot of shrinking and shattering under the scope. Andi G Andrea Grantham, HT (ASCP) Senior Research Specialist University of Arizona Cellular and Molecular Medicine Histology Service Laboratory P.O.Box 245044 Tucson, AZ 85724 algra...@email.arizona.edumailto:algra...@email.arizona.edu Tel: 520.626.4415 Fax: 520.626.2097 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Project DNA-Hereditary Cancers
FYI to my Histonet peeps. New developments in Hereditary Cancer. I am so proud to be associated with such a progressive GI doctor who has a passion towards genetic cancer solutions! Check out what the CEO of our GI practice/pathology laboratory, Dr. Daniel Luba's passion is on this website. He is the Chairman of the Board for project DNA. http://theprojectdna.com/about/ With warmest regards, Akemi Akemi Allison BS, HT/HTL (ASCP) Pathology Manager Monterey Bay GI Consultants Laboratory 23 Upper Ragsdale Drive, Suite 200 Monterey, CA 93940 Email: aalli...@montereygi.com Tele: (831) 375-3577 X117 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: ISH preparation
Leica's Thermobrite Elite? http://www.leicabiosystems.com/pathology-imaging/cytogenetics/details/product/leica-thermobrite-elite/ Joana Moreira Supervisor - Anatomical Pathology Department of Pathology Sidra Medical Research Center PO Box 26999 | Doha, Qatar Direct Line +974-4404-2036 jmore...@sidra.org | www.sidra.org Message: 6 Date: Wed, 25 Jun 2014 08:38:29 -0400 From: Emily Brown talulahg...@gmail.com Subject: Re: [Histonet] ISH preparation To: jennifer.john...@genzyme.com Cc: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: CAP=xx1wxtnatbywkh7ekwz0ktkmfjvbv+422yy6xjhy_6se...@mail.gmail.com Content-Type: text/plain; charset=UTF-8 That may be what you have to do, as the first day of ISH needs to be RNase free! When I do them, it is 10 slides at a time and we have dishes and a lab bench set aside for RNase free procedures only. Obviously this is not feasible in all workplaces! I would just think about it logically. Use gloves when you can, always. Can you keep the hybridizing part RNase free? I don't know what machines you use for this--we do process the slides manually in glass dishes and then hybridize in a slide container and oven kept for RNase free purposes. After hybridization, you don't really need to be too careful. Now I'm wondering if there's some sort of magical machine that does ISH! 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 Mon, Jun 23, 2014 at 2:06 PM, jennifer.john...@genzyme.com wrote: Hi Folks! I was hoping to get a little input about preparing samples for ISH. We currently RNAse-away our tools at the time of collection, toss the tissues into formalin, process normally and then cut them using RNAse away cleaned microtomes/forceps on a bath of DEPC treated water (in a cleaned water bath). How do other labs prepare paraffin embedded samples? Do you use special reagents - formalin? Do you clean the processor and use treated reagents there too? Do you wash the slides in DEPC treated water? I am trying to figure out how far we need to go here. We are a high throughput lab and we deal with all kinds of tissues and I don't want to have to take down a processor and keep it RNAse free if I don't have to! I will run the experiments and test different methods, but I was hoping some of you could share your wisdom and give me a place to start. Thanks, JJ Jennifer Johnson Staff Scientist Genzyme Corp. Department of Pathology 5 Mountain Road Framingham, MA 01701-9322 Phn - 508-271-3610 Fax - 508-872-9080 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Disclaimer: This email and its attachments may be confidential and are intended solely for the use of the individual to whom it is addressed. If you are not the intended recipient, any reading, printing, storage, disclosure, copying or any other action taken in respect of this e-mail is prohibited and may be unlawful. If you are not the intended recipient, please notify the sender immediately by using the reply function and then permanently delete what you have received. Any views or opinions expressed are solely those of the author and do not necessarily represent those of Sidra Medical and Research Center. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
