[Histonet] anti-mouse NK marker
Hi, Does anyone have a good anti-mouse NK cell marker for frozen mouse tissues? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] histology tracking systems for pharma or contract lab
Hi, My company is considering a new tracking system for our histology studies. This system needs to track everything from trimming, decal, embedding, cutting, routine and molecular pathology/staining. It also needs to be able to accommodate complex study designs (multiple animals, groups, necropsy timepoints, etc). I am interesting in hearing from people in other pharma/biotech/CROs to see what they are using to capture this information. Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] alternates to cryojane
Hello eveyone, Are there any other tape transfer systems for the cryostat other than the cryojane? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] eosin in the processor
Hi Everyone, Years ago, my lab used to put eosin in the processor to lightly tint the smaller mouse tissues. I can't remember which station we put it in (I think it was the 2nd 100% ethanol). Also, back then my lab didn't do any IHC; will the eosin affect any IHC that might be done (I am guessing no, but I want to be sure). Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] CD31 for FFPE IHC on Mouse
I second the Dianova antibody - we have had very good luck with it. Standard citrate HIER works for us. Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Patsy Ruegg pru...@ihctech.net To: 'Mark Elliott' mark.elli...@hli.ubc.ca; histonet@lists.utsouthwestern.edu Sent: Sunday, October 21, 2012 11:20 AM Subject: RE: [Histonet] CD31 for FFPE IHC on Mouse Rat anti mouse cd31 from Dionova is the way to go it is better than all the others. Patsy Ruegg, HT(ASCP)QIHC Ruegg IHC Consulting, LLC 40864 Arkansas Ave Bennett, CO 80102 Phone: 303-644-4538 Fax: 720-859-4110 pru...@ihctech.net -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mark Elliott Sent: Friday, October 19, 2012 3:45 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] CD31 for FFPE IHC on Mouse Amy Can you please share the responses you got with the rest of us Thanks Mark Message: 8 Date: Thu, 18 Oct 2012 16:21:55 -0400 From: Amy Porter port...@msu.edu Subject: RE: [Histonet] CD31 for FFPE Immunohistochemistry on Mouse Model To: 'Amy Porter' port...@msu.edu,'Histonet' histonet@lists.utsouthwestern.edu Message-ID: 003101cdad6e$3792fc80$a6b8f580$@edu Content-Type: text/plain;charset=US-ASCII Thanks to all for responses..looks like most roads lead to once place which is spectacular!! Just what I needed. Amy -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amy Porter Sent: Thursday, October 18, 2012 3:36 PM To: 'Histonet' Subject: [Histonet] CD31 for FFPE Immunohistochemistry on Mouse Model Anyone out there have CD31 working well on FFPE samples for Mouse Samples?? I know this might be a long shot, however I haven't looked for anything on this in quite awhile. Amy S. Porter, HT(ASCP) QIHC Michigan State University Investigative HistoPathology Laboratory William S. Spielman, Ph.D. - Director Patricia K. Senagore, M.D. - Consulting Pathologist Department of Physiology / Human Pathology Biomedical Physical Sciences Building 567 Wilson Road - Room 2133 East Lansing, MI 48824-3320 Phone: 517-884-5026 Fax: 517-432-1368 port...@msu.edu http://www.humanpathology.msu.edu/ ***CONFIDENTIALITY NOTICE*** This electronic message and any attachments are intended only for the use of the addressee and may contain information that is privileged and confidential. Any dissemination, distribution or copying of this communication by unauthorized individuals is strictly prohibited. If you have received this communication in error, please notify the sender immediately by reply e-mail and delete the original and all copies from your 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] human vimentin in mouse tissue
Greetings! I want to stain for human vimentin in mouse tissues (human fibroblasts). I many years ago, I used a biotinylated V9 clone for just this purpose and it worked great. I tried the biotiylated V9 clone from abcam, but I am not getting any staining at all. Any suggestions? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] endogenous peroxidase in eosinophils
Anyone have tips for quenching endogenous peroxidase in eosinophils? Our standard px block is not doing the job (Biocare Peroxidazed-1). Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] fibrin IHC on FFPE mouse tissue
Is there an antibody out there that will stain for fibrin on FFPE mouse tissues; been searching but haven't found one. Does everyone just do a special stain when looking for fibrin? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning
As long as you don't need to use them for RNA analysis, the easiest thing to do is just rub your finger (without glove) across the block (very briefly) and then take the section. This will probably do the trick and hydrate it just enough to make a difference. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Kasai, Miki (NIH/NCI) [E] kas...@mail.nih.gov To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, February 27, 2012 1:34 PM Subject: [Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning We are trouble-shooting cryosectioning mouse lung tissue. Often the lung section tears or breaks apart during sectioning. In the past, if the lung section is proving difficult to section, we take the OCT-embedded tissue and re-embed it back into OCT (basically put fresh OCT into the original mold and then place the OCT block with the tissue back into the mold such that the exposed tissue is covered back with OCT). This is then placed back in our -80°C. When sectioning the next day, the tissue is often easier to section. One person in our lab tried to resolve the problem by brushing a little bit of sterile water onto the tissue when sectioning. This appeared to hydrate the tissue and it sectioned better. However, we weren't sure if this was a good idea or not. Any feedback would be greatly appreciated. For background purposes our lung tissue are processed several ways: 1. Lungs are perfused with PBS, tissue extracted from mouse, placed in PFA/sucrose for several hours and then embedded in OCT. 2. Lungs are perfused with PBS, inflated gently with OCT/PBS (3:1), placed in PFA/sucrose for several hours and then embedded in OCT. 3. Lungs are perfused with PBS, inflated gently with OCT/PBS (3:1), embedded in OCT and frozen by immersion into liquid nitrogen (just the bottom half of the mold is lowered into LN). Much appreciation, Miki Kasai Biologist Pediatric Oncology Branch NCI, NIH CRC, 1W Rm. 1-3-888 10 Center Drive Bethesda, MD 20892 (301) 496-2318 ___ 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] Hydrating Frozen Tissues embedded in OCT for Sectioning
Also, make sure that the block sits in the cryostat for a while to acclimate to the temperature. The cutting temp for lung (correct me if I'm wrong here) is probably about -20; the block should be the same temperature as the cryostat. Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Colleen Forster cfors...@umn.edu To: Kim Merriam kmerriam2...@yahoo.com Sent: Monday, February 27, 2012 1:55 PM Subject: Re: [Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning Yep, the sample is too cold. Rubbing your finger across even with a glove (just linger a bit longer) will help alot. Colleen Forster U of MN On 2/27/2012 12:47 PM, Kim Merriam wrote: As long as you don't need to use them for RNA analysis, the easiest thing to do is just rub your finger (without glove) across the block (very briefly) and then take the section. This will probably do the trick and hydrate it just enough to make a difference. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Kasai, Miki (NIH/NCI) [E]kas...@mail.nih.gov To: histonet@lists.utsouthwestern.eduhistonet@lists.utsouthwestern.edu Sent: Monday, February 27, 2012 1:34 PM Subject: [Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning We are trouble-shooting cryosectioning mouse lung tissue. Often the lung section tears or breaks apart during sectioning. In the past, if the lung section is proving difficult to section, we take the OCT-embedded tissue and re-embed it back into OCT (basically put fresh OCT into the original mold and then place the OCT block with the tissue back into the mold such that the exposed tissue is covered back with OCT). This is then placed back in our -80°C. When sectioning the next day, the tissue is often easier to section. One person in our lab tried to resolve the problem by brushing a little bit of sterile water onto the tissue when sectioning. This appeared to hydrate the tissue and it sectioned better. However, we weren't sure if this was a good idea or not. Any feedback would be greatly appreciated. For background purposes our lung tissue are processed several ways: 1. Lungs are perfused with PBS, tissue extracted from mouse, placed in PFA/sucrose for several hours and then embedded in OCT. 2. Lungs are perfused with PBS, inflated gently with OCT/PBS (3:1), placed in PFA/sucrose for several hours and then embedded in OCT. 3. Lungs are perfused with PBS, inflated gently with OCT/PBS (3:1), embedded in OCT and frozen by immersion into liquid nitrogen (just the bottom half of the mold is lowered into LN). Much appreciation, Miki Kasai Biologist Pediatric Oncology Branch NCI, NIH CRC, 1W Rm. 1-3-888 10 Center Drive Bethesda, MD 20892 (301) 496-2318 ___ 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] Cytology CSF Cell Pellets made from Histogel
I work in a research lab, but I use histogel all the time to make FFPE blocks of cell culture material. Here is my procedure: 1. you need about 5X10^ cells per pellet 2. spin cells @ 2000 rpm for 5 minutes in a 50 ml conical tube 3. aspirate and resuspend in 15ml NBF and fix overnight @ RT 4. spin @ 2000 rpm for 5 minutes and aspirate the NBF 5. microwave the histogel (thermo #HG-4000-012) for 15-20 seconds (loosen cap before microwaving) 6. histogel should now be at liquid for and about 60C 7. resuspend cell pellet in about 350ul of histogel 8. place cell pellet in freezer or on ice for about 20 minutes to solidify 9. using spatula, remove histogel pellet and place into cassette 10. post-fix in NBF for 2 hours (this step is very important); histogel will dissolve in processor if not fixed 11. process as usual Good luck! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Turner, Leandra lturn...@seton.org To: histonet@lists.utsouthwestern.edu Sent: Tuesday, February 21, 2012 12:26 PM Subject: [Histonet] Cytology CSF Cell Pellets made from Histogel Hello Everyone, I am trying to find out a few of things about making cell pellets on cerebrospinal fluids. I would like to know: 1. If anyone has ever made cell pellets from CSF's using Histogel and has any tips or procedures they could share? 2. How to process the CSF pellets made with Histogel, do we need a routine or stat process? (we use a Sakura Tissue Tek) 3. Can you do IHC staining on the pellets? Thank you for any and all help in advance. Leandra Turner, HT (ASPC)CM ___ 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] Cytology CSF Cell Pellets made from Histogel
I forgot to mention, we prepare these specifically for IHC; so yes, you can do IHC on them. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Kim Merriam kmerriam2...@yahoo.com To: Turner, Leandra lturn...@seton.org; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Wednesday, February 22, 2012 8:21 AM Subject: Re: [Histonet] Cytology CSF Cell Pellets made from Histogel I work in a research lab, but I use histogel all the time to make FFPE blocks of cell culture material. Here is my procedure: 1. you need about 5X10^ cells per pellet 2. spin cells @ 2000 rpm for 5 minutes in a 50 ml conical tube 3. aspirate and resuspend in 15ml NBF and fix overnight @ RT 4. spin @ 2000 rpm for 5 minutes and aspirate the NBF 5. microwave the histogel (thermo #HG-4000-012) for 15-20 seconds (loosen cap before microwaving) 6. histogel should now be at liquid for and about 60C 7. resuspend cell pellet in about 350ul of histogel 8. place cell pellet in freezer or on ice for about 20 minutes to solidify 9. using spatula, remove histogel pellet and place into cassette 10. post-fix in NBF for 2 hours (this step is very important); histogel will dissolve in processor if not fixed 11. process as usual Good luck! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Turner, Leandra lturn...@seton.org To: histonet@lists.utsouthwestern.edu Sent: Tuesday, February 21, 2012 12:26 PM Subject: [Histonet] Cytology CSF Cell Pellets made from Histogel Hello Everyone, I am trying to find out a few of things about making cell pellets on cerebrospinal fluids. I would like to know: 1. If anyone has ever made cell pellets from CSF's using Histogel and has any tips or procedures they could share? 2. How to process the CSF pellets made with Histogel, do we need a routine or stat process? (we use a Sakura Tissue Tek) 3. Can you do IHC staining on the pellets? Thank you for any and all help in advance. Leandra Turner, HT (ASPC)CM ___ 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] Immunofluorescence staining/minimizing background staining
Hi, What fluorochromes are you using? There is a lot of autofluorescence in the FITC channel. Have you looked at an unstained tissues under the scope with each filter that you need, that may give you a clue as to where your background is coming from. In addition to an unstained slide, I suggest eliminating the primary to see if your secondary is sticking to the tissues. Are you doing single-color or double stains? If you are getting a lot of autofluorescence with one fluorochrome, I would suggest trying a different one. We use alexafluur-647 (far red) a lot because is it fairly clean (ie - very little autofluorescence in that channel) Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Kasai, Miki (NIH/NCI) [E] kas...@mail.nih.gov To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, January 30, 2012 4:50 PM Subject: [Histonet] Immunofluorescence staining/minimizing background staining Hi, We are performing some immunofluorescence staining on mouse lung tissue. We are getting some nice positive staining with some of our initial antibodies (procollagen, cytokeratin). We would like to minimize the amount of background staining we are getting. We are titering our primary antibodies to find out optimal Ab concentration as well as the secondary conjugate Ab with the fluorophore of interest. We use donkey serum for general blocking. Any other suggestions? Much appreciation, Miki ___ 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: Interview Questions
I had a great question asked of me when I was interviewed for my current position: Tell me of a time that you had to rescue some tissue; what happened to it that ruined it and what did you do to fix it? A great question for a histologist. People with experience in the field should have several stories related to this question (especially in research, when you have PIs collecting tissue for you that often have no clue what they are doing)!!! Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Burton, Lynn lynn.bur...@illinois.gov To: Breeden, Sara sbree...@nmda.nmsu.edu; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Wednesday, January 25, 2012 10:42 AM Subject: [Histonet] RE: Interview Questions I don't have any ideas but I am sorry to hear you are leaving. Good Luck with your future. Lynn Burton Lab Assoc I Animal Disease Lab Galesburg, Il 309-344-2451 From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Breeden, Sara [sbree...@nmda.nmsu.edu] Sent: Wednesday, January 25, 2012 9:37 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Interview Questions Okay, My People - I will be one of the interviewers for locating my replacement). I've not been this fortunate before and I do know there are questions one cannot ask so that's not an issue. What I'd like to know is what I SHOULD ask. This position is fairly straightforward - basic veterinary histology with nothing significantly challenging (but with that potential). What would YOU want to know about a candidate that would convince you that this person was The One? I need questions with meat to them. Your suggestions will be much-ly appreciated. Gracias! Sally Breeden, HT(ASCP) New Mexico Department of Agriculture Veterinary Diagnostic Services 1101 Camino de Salud NE Albuquerque, NM 87102 505-383-9278 (Histology Lab) ___ 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] neutrophil marker for FFPE mouse tissue
Hello! What antibodies are people using to stain for neutrophils in FFPE mouse tissue? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Efficacity of fluorochrome in Immunofluorescence
You are likely getting some cross-reactivity with your secondary antibodies (one of them may be crossing to the other primary). We always run a battery of control slides when validating a double-IF stain. Run these controls and you will likely figure out where the problem is: Slide Set Antibodies 1 Primary #1/Secondary#1 2 Primary#2/Secondary #2 3 Isotype#1/Secondary #1 4 Isotype #2/Secondary #2 5 Primary #1/Primary #2/Secondary #1/Secondary#2 6 Isotype #1/Isotype #2/Secondary#1/Secondary #2 7 Primary #1/Isotype #2/Secondary #1/Secondary#2 8 Isotype #1/Primary#2/Secondary #1/Secondary #2 9 NoPrimary (diluentonly)/Secondary #1 10 No Primary(diluentonly)/Secondary #2 11 No Primary (diluentonly)/Secondary #1/Secondary #2 12 Diluent only (or nothing) 13 DAPI only Best of luck! Kim, Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Natalia Fernandez nunu...@hotmail.com To: histonet@lists.utsouthwestern.edu Sent: Tuesday, January 24, 2012 4:45 AM Subject: [Histonet] Efficacity of fluorochrome in Immunofluorescence Hi everybody! Maybe you can help me. I'm trying to do immunofluorescences to see the colocalisation between two proteins (PSD + Oligomeres) using Oregon Green (goat anti mouse) and Texas Red (goat anti rabbit). But my probleme, is that the analysis of the slides reveals too much colocalisation. I mean, all seems yellow, as if both fluorochromes show the same thing. It's the first time I do fluorescence, so I don't know where could come the problem. I tried to do a simple staining, 1 slide with my first antibody and its fluorochrome, and the second slide with the other first antibody with its fluorochome and then check the result with the filter red and green. But it's strange because I see both color as if I had put both fluorochromes (second antibodies). Has somebody already had this problem? What can I do to resolve it? Thank you very much for your help. Natalia ___ 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] proximity of proteins for co-localization via IF staining
Hi Everyone, Does anyone know how close in proximity 2 proteins need to be for them to show up as co-localized via immunofluorescence staining? I am trying to compare routine 2-color IF vs. the Proximity Ligation Assay (which will not show co-localization unless the proteins are withing 40 nM of each other). Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] brain sections cut on Cryo Jane apparatus are falling off slides during ethanol series for a cresyl stain
I was having similar issues with frozen rat and human cartilage with the cryojane. After a lot of trial and error; I have found that the 4X slides and air-drying overnight has worked extremely well for us. The tissues never fall off any more! Good luck, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Connolly, Brett M brett_conno...@merck.com To: Douglas M Burns dmbur...@gmail.com; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Thursday, December 8, 2011 9:46 PM Subject: RE: [Histonet] brain sections cut on Cryo Jane apparatus are falling off slides during ethanol series for a cresyl stain Doug, Hopefully you are using the CryoJane slides (not the subbed slides) and the UV glue activator and keeping both slides and tape cold in the cryostat. Be aware that you can get CryoJane slides with different amounts of adhesive (0.5-4x). I would suggest going to the 4x slides. When peeling the tape off I pull it slowly and evenly almost back on itself at an acute angle- this greatly reduces the incidence of the section coming off with the tape... Haven't had a problem with section detachment in ethanols. Brett Brett Connolly, Phd Merck Research Labs West Point, PA From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Douglas M Burns [dmbur...@gmail.com] Sent: Thursday, December 08, 2011 5:29 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] brain sections cut on Cryo Jane apparatus are falling off slides during ethanol series for a cresyl stain Hello, Histo-users, Follow-up to our original posting about fixed-brain cryosections falling off of subbed slides. We fished out our Cryo-Jane apparatus, and using this apparatus has resulted in a great improvement in sections and getting sections stuck onto the slides. This works pretty well until we start using the ethanol series to dehydrate and delipidate before a Crystal violet stain for Nissl bodies. (That is, for acqueous steps everything seems to stay glued to the slide; however, once ethanols are involved pieces start to come off.) To summarize, we are trying to go from cut blocks of paraformaldehyde-fixed brain infused with sucrose and then frozen at -80. We take them out, unfrozen, and cut them on a Leica cryostat. Early attempts with subbed slides did not work reliably, so we have turned to 'Jane. Jane is slightly slower than a brush, and seems to work fairly well. However, we have had trouble with the tape pulling parts of sections off of the glued slides. When we introduce the nonacqeous solvents, our problems become more obvious. We have not so far gone to a post-fix, because we wanted to simplify the overall process. Could this be the mistake? We want to use IHC for c-Fox and possibly orexin - and similar - but we want cresyl-stain to locate positively the area of interest in the cut block. Any suggestions? Also, are there any 'Jane enthusiasts out there? thank you in advance --- Doug Burns, Kansas City ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your 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
[Histonet] counterstain for fast red
Hi Everyone, We are currently running some alk phos staining protocols with a fast red chromogen. My pathologist would like to use something other than Hematoxylin as a counterstain. What does everyone recommend? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] ACD RNAscope
I have been trying to get it to work using their actin (and control probes) probe on some routine FFPE cell pellets that I had, and I have not had any luck at all with it. Truly, the kit is a breeze to use, but I can't get the darn thing to work. I have had some very modest success with their kit on frozen sections. I have shipped them some of my slides and they have agreed to test them in their lab. Let me know if you have any success! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Louise Renton louise.ren...@gmail.com To: Histonet Histonet@lists.utsouthwestern.edu Sent: Tuesday, August 30, 2011 5:13 AM Subject: [Histonet] ACD RNAscope Hi all - is anyone using the above system for RNA detection in FFPE tissues - it sounds almost too good to be true? -- 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] instructions for steaming slides
We use a black and decker rice steamer fairly routinely. It has the ability to steam for a total of 75 minutes, although it takes a good 15 of them to get up to temperature. I like it becasue the temperature is nice and even and it is very easy to do. 1. add water to the bottom of the steamer and turn it on (I turn the dial all the way to 75 minutes) 2. preheate the HIER solution in the microwave (90-120 seconds for a tissue-tek staining bucket is fine) 3. add slides to the solution and place into the top compartment of the steamer 4. steam for desired time (in tests that I did many years ago, most antibodies required a minimum of 45 minutes in the steamer) 5. you can check the temperature of your retrieval solution, it should be about 100C 6. once done, remove, cool, rinse in water and proceed with your staining Good luck! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Kant, H.J.G. van de (Henk) h.j.g.vandek...@uu.nl To: 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu Sent: Thursday, August 25, 2011 1:49 AM Subject: [Histonet] instructions for steaming slides Dear All, Can somebody help me with instructions (protocol) for steaming slides in a domestic ricecooker or vegetable steamer. Head induced antigen retrieval method is with sodium citrate 0.01 M pH 6.0. Kind regards, Henk van de Kant | Utrecht University | Faculty of Science | Department of Pharmaceutical Sciences | Division of Pharmacology David de Wied building (room 2.21) | Universiteitsweg 99 | 3584 CG Utrecht | The Netherlands ___ 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] hospitality events at NSH
Anyone heard anything about hospitality events at the NSH? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Reorienting frozen tissue
Whenever I have to reembed an OCT block, I leave it out at RT for just a couple of minutes to let it that a little bit. Just take a razor blade and cut out the tissue (leaving some OCT around it); re-oreint it in a mold with RT OCT and then freeze it. Don't freeze it too fast, you want the old OCT to melt a little bit with the new OCT, that way you will have a block without any seams in it. Good luck! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Daniela Bodemer daniela.bode...@mcri.edu.au To: histonet@lists.utsouthwestern.edu Sent: Tuesday, August 2, 2011 7:44 PM Subject: [Histonet] Reorienting frozen tissue Hi all, I would like to know if it is possible and how to reorient or reembedd human tissue, that has been frozen at -80C in OCT. Many thanks, Daniela Bodemer Research Assistant Surgical Research, Infection and Immunity Murdoch Childrens Research Institute The Royal Children's Hospital Flemington Road Parkville Victoria 3052 Australia T 03 9345 5930 T (03 9345 4116) E daniela.bode...@mcri.edu.au mailto:firstname.surn...@mcri.edu.au www.mcri.edu.au http://www.mcri.edu.au/ This e-mail and any attachments to it (the Communication) are, unless otherwise stated, confidential, may contain copyright material and is for the use only of the intended recipient. If you receive the Communication in error, please notify the sender immediately by return e-mail, delete the Communication and the return e-mail, and do not read, copy, retransmit or otherwise deal with it. Any views expressed in the Communication are those of the individual sender only, unless expressly stated to be those of Murdoch Childrens Research Institute (MCRI) ABN 21 006 566 972 or any of its related entities. MCRI does not accept liability in connection with the integrity of or errors in the Communication, computer virus, data corruption, interference or delay arising from or in respect of the Communication. P Please consider the environment before printing this email __ This email has been scanned by the MessageLabs Email Security System. For more information please visit http://www.messagelabs.com/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] Mouse vs. Human cells
Years ago, we used to distinguish human fibroblasts in mouse tissue with vimentin; which stains human cells quite nicely, but does not cross to mouse. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Carl Postenka cposte...@hotmail.com To: sdys...@mirnarx.com; histonet@lists.utsouthwestern.edu Sent: Tuesday, August 2, 2011 11:16 AM Subject: RE: [Histonet] Mouse vs. Human cells We've had succes identifying human cells in a mouse background using an antibody against human mitochondria (Neomarkers cat#MS-1372P). The primary is a monoclonal mouse anti-human, so we use a Dako ARK kit (#K3954) to eliminate the mouse on mouse background. HIER using citrate pH=6.0. H2O2 block. Primary diluted 1/100 (through ARK kit), for 20min @ R/T. -- Carl PostenkaLondon Regional Cancer Program Date: Mon, 1 Aug 2011 12:55:54 -0500 From: sdys...@mirnarx.com To: Histonet@lists.utsouthwestern.edu CC: Subject: [Histonet] Mouse vs. Human cells Hello Histo-hotties! Question: We are working with xenograft tumors in mice. The tumor cells are human in origin. I am trying to come up with some kind of stain that will stain all the mouse cells and none of the human cells. Just to be able to determine if any of the human tumor cells are in a normal looking say liver. I was thinking maybe Ki-67? It doesn't have to be any specific marker, just to be able to see a really blue mouse organ, and then if there are human cells of any type in the organ light up that one cell. Thanks Sarah Goebel-Dysart, BA, HT(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 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] RNAscope ISH technology
Happy Friday! Does anyone have experience using RNAscope ISH technology on FFPE tissues? http://www.acdbio.com/whats_rnascope.html Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] molecular path email listserv
Hi Everyone, Does anyone know of an email listserv similar to this one for molecular pathology? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Biocare Intelipath
I second Karen's comments about Biocare and the Intellipath. I work in an early discovery research pathology lab at a large biotech firm. We do not have a standard set of stains that we run (we are testing new ones all the time), so the open-ness of the instrument was key. You can use any antibody or detection system (we use antibodies from many sources, including those made in-house), but we tend to use Biocare's detection reagents for chromogenic staining. We also do all of our IF staining on the machine. I am a long-time customer of Biocare's and I even got to beta-test the Intellipath instrument a few years ago. They are by far one of the best companies I have ever worked with, they are super customer-oriented and the service engineers come quickly, if your machine needs to be fixed. I would defintely get a demo into your lab to see if it fits your needs. Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Karen Lahti ka...@gateslinger.com To: Sheila Adey sa...@hotmail.ca; histonet@lists.utsouthwestern.edu Sent: Mon, May 9, 2011 12:47:22 PM Subject: RE: [Histonet] Biocare Intelipath Sheila, I have been a long time Biocare user, as well as many years of experience with just about every IHC company/instrument on the market today. I have had a fair share of issues with each. I am amazed continuously at the attention and care I am given from the whole Biocare company. When I call with even a minor maybe problem, I do feel as if I am their only customer. I get immediate response. The IntelliPath instrument does require more tech time to prepare the slides which I resisted from the very beginning. I have learned the flexibility this has brought to validation and overall quality of staining. The antibodies and detection are exceptional in comparison to other vendors. The detection coupled with the instrument enabled our lab to drastically reduce the cost of each stain and keep a high level of quality that is reproducible from day to day. Karen Karen D. Lahti, HT(ASCP)QIHC, MLT(AMT) Histology Supervisor Arizona Digestive Health 1300 N. 12th Street #550 Phoenix, AZ 85006 480-236-6559 cell 602-687-7218 office -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Sheila Adey Sent: Sunday, May 08, 2011 3:57 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Biocare Intelipath Hello Netters We are considering an Intelipath. I'd like some opinions on the instrument from users out there. Thanks in advance!!! Sheila ___ 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] Senior Histologist/Lab Manager position at AstraZeneca in Waltham, MA
Hello all, Below is a great job opportunity for a senior histologist/lab manager at AstraZeneca in Waltham, MA. I am posting this for a colleague, so please do not email me if you are interested. Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA http://jobs.astrazeneca.com/jobs/39-histotechnologistscientist Here is the job description: Histotechnologist/Scientist Reference Number 601367 Date Added 18/02/2011 Category Research and Non-Clinical Develo... Company AstraZeneca Country United States Location Massachusetts (Waltham) Incumbent lead a small Histology Laboratory operating in a discovery and safety assessment environment and assist in the conduct of experiments/studies that will include investigative, problem-solving, and pre-development studies in support of research teams. Major Responsibilities: * As a working supervisor, provide day-to-day management of the Histology Laboratory and Necropsy Suite * Provide leadership, supervision, and mentoring to histology team members * Work closely with the histologists, pathologists, research scientists, and toxicologists within Safety Assessment US and Global Safety Assessment regarding study plan development, completion and reporting * Ensure smooth operations and timely completion of necropsies, trimming, embedding and HE staining, quality control, and other histology procedures such as frozen sections, immunohistochemistry (IHC) and in situ hybridization (ISH) * Develop new histology, necropsy, and IHC procedures as necessary * Perform large and small animal necropsies * Ensure Histology Laboratory and Necropsy Suites have appropriate standard operating and safety procedures in place and that staff are compliant * Serve on some studies as the histologist responsible for study specific support (Histology Study Coordinator) * Be familiar with and provide local management of histology and pathology-related data systems (e.g., PathData®) * Maintain adequate and appropriate supplies for the histology laboratory and necropsy suite Requirements Qualifications: Skills, Knowledge/Education/Experience * Bachelor of Science (BS), MS desirable * Certified Histology Technician (HT/HTL) * Minimum 7 years industry experience * Laboratory management experience * Knowledge and familiarity with histology laboratory equipment and all phases of operations of a histology laboratory * Experience with large and small animal necropsy * Experience with special stains, plastic-embedding and molecular techniques such as immunohistochemistry * Experience in working with and managing Standard Operating Procedures (SOPs) * Knowledge of Good Laboratory Practices (GLP) standards, laboratory safety procedures, and chemical hygiene management * Enthusiastic, self-motivated, leadership and mentoring skills * Excellent communication skills (written and oral) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] TRAP staining kit
Hi All, Does anyone know if there is a TRAP staining kit available for histology (and who sells it)? I have found a bunch of them on google, but they are all for cell culture. Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] OFF-TOPIC - stuff to do in New Orleans
Hi Guys, I am sure that there are New Orleans histonetters out there. I am going there in a couple of weeks for a few days with the kids (boys, age 9 and 14). I was wondering if anyone could suggest some good places to go, for both kids and grownups (we are going with other families and have built-in babysitters). Thanks a bunch, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] fibroblast marker in mouse tissue
Hi All, What is everyone using to stain for fibroblasts in frozen mouse tissue? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] cryojane problems
Brett- I have been using the 4X slides, but they don't seem to help. I dont have this problem with the cartilage alone, but when there is bone attached, it is a nightmare. Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Connolly, Brett M brett_conno...@merck.com To: Kim Merriam kmerriam2...@yahoo.com; Histonet histonet@lists.utsouthwestern.edu Sent: Tue, October 26, 2010 9:41:18 AM Subject: RE: [Histonet] cryojane problems Kim, I would recommend using their '4x' slides - they have more adhesive on them. I don't think flashing more than once has any benefit. Brett Brett M. Connolly, Ph.D. Molecular Imaging Team Leader Merck Co., Inc. PO Box 4, WP-44K West Point, PA 19486 brett_conno...@merck.com T- 215-652-2501 F- 215-993-6803 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Kim Merriam Sent: Tuesday, October 26, 2010 9:32 AM To: Histonet Subject: [Histonet] cryojane problems Hi All, I have been using the cryojane to cut human articular cartilage, with excellent results. Now I need to cut frozen sections of rat articular cartilage that contain some underlying bone. I am getting nice sections, but when I peel the tape off the slide, a bunch of my tissue is staying on the tape. I have tried increasing the number of flashes (to a whopping 10!), hoping that this will help adhere the sections, but it hasn't. Any tips for me? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] cryojane problems
Hi All, I have been using the cryojane to cut human articular cartilage, with excellent results. Now I need to cut frozen sections of rat articular cartilage that contain some underlying bone. I am getting nice sections, but when I peel the tape off the slide, a bunch of my tissue is staying on the tape. I have tried increasing the number of flashes (to a whopping 10!), hoping that this will help adhere the sections, but it hasn't. Any tips for me? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] special stain for H. billis
Good morning everyone, Does anyone know of a special stain that is specifically for H. billis. I don't know much about bacteria, so I am not even sure which bacterial stain would work on this. Any comments would be appreciated. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] human on human IHC
Hi, Does anyone know of a human-on-human IHC kit similar to the mouse-on-mouse kits that are available? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] tungsten knives for cryostat
Hi All, I am in need of some disposable tunsten knives for my cryostat. It currently has a low-profile blade holder on it, but I can't seem to find any disposable tungsten knives that are low-profile. Does anyone know if they are available? If not, I can just buy some high-profile ones and get a new adaptor for my knife holder. What company makes the best tungsten knives? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] off-topic cryosectioning as security check word
Hi All, I thought you all would get a kick out of this. I was ordering some tickets on ticketmaster and look at the word that popped up for the security check - it was cryosectioning! I managed to get a screenshot of it; it is attached. We have infiltrated main-stream culture! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Chilling Tray for Blocks
we use something like this: https://www.vwrsp.com/catalog/product/index.cgi?catalog_number=414004-256inE=1highlight=414004-256 we keep several sizes in the freezer, depending on how many blocks we need to cut. The nice thing is that it is cheap and you can hydrate your blocks at the same time you are chilling them. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Sherwood, Margaret msherw...@partners.org To: Jay Lundgren jaylundg...@gmail.com; Laurie Colbert laurie.colb...@huntingtonhospital.com Cc: histonet@lists.utsouthwestern.edu Sent: Wed, June 30, 2010 4:35:53 PM Subject: RE: [Histonet] Chilling Tray for Blocks We actually use a styrofoam box (used for shipping) and it stays cold longer with very little melting. We keep a couple in the -20 freezer. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jay Lundgren Sent: Wednesday, June 30, 2010 4:01 PM To: Laurie Colbert Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Chilling Tray for Blocks Fill a Tupperware container with water, pop it in the freezer, and tomorrow you will have a portable block chiller, with the blocks at a perfect level for your needs, for 1/100th the price. Not trying to be a smarta$$, but Jay A. Lundgren, M.S., HTL (ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. ___ 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] cryojane question
Happy Friday! We recently purchased a cryojane for sectioning frozen human and rat cartilage. I am having some issues with my cartilage sections. I am using the 4X slides with 2 flashes. The sections look beautiful, until I put them into buffer to do staining. I am getting folds and the sections are lifting off during IHC staining. I am staining either by hand (on flat trays, old-fashioned style) or in the sequenza racks. Luckily, the cartilage is not not coming off the slides completely, so I have been able to get the data I need. I was wondering if anyone had any tips for me to prevent the folds or the lift-off. Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] ISH on histogel cell pellets
Hi Everyone, We have some cells that we have processed and embedded in histogel (thanks to everyone for the protocols that were sent to me a couple of weeks ago). We are planning to do IHC and ISH on these pellets. Will the histogel interfere with the ISH procedure at all? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Histodeck Flash Cards
Hi Laurie, I purchased them because I am thinking about taking the HTL in the next year or so. The are pretty nice, they are divided up into categories (fixation, processing, etc) and each category has a different color edge on the card. They have questions on the front with the answers and explanations on the back. They were pretty expensive (75-80, I think), but would probably help out a lot when studying for an exam (a non-histologist could ask the questions and help you study)! I will probably enlist my 9 year old, who gets a kick out of this kind of thing. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: lau...@conxis.com lau...@conxis.com To: histonet@lists.utsouthwestern.edu Sent: Tue, June 8, 2010 11:20:46 AM Subject: [Histonet] Histodeck Flash Cards Hi Everyone, I was wondering if anyone out there has used the Histodeck flash cards to study for the HTL exam? Just curious. Thanks, Laurie *** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] ICC slide storage
Hi Sarah, I used to make a ton of cytospins. I would air-dry them, fix them in whatever (ethanol or acetone/ethanol), air-dry them again and then wrap the slides in foil, box them up and store @ -80C. The slides lasted for months! When taking them out to use for staining, be sure to allow the wrapped slides to acclimate to RT before you use them. Happy staining! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: sgoe...@xbiotech.com sgoe...@xbiotech.com To: histonet@lists.utsouthwestern.edu Sent: Tue, May 18, 2010 2:36:56 PM Subject: [Histonet] ICC slide storage So, again...I'm new to this ICC staining. I got a epindorf = tube of cells (and a PBMC), I made cytospins from them and then fixed them in acetone, then 100 alcohol, then rehydrated them in PBS. The f irst day I did the spins the slides looked great, you could definately see = the monocytes. Then the days that have followed the cells have become= lysed, and there is gradoo all over the place. I think that it pro= bably has something to do with the fact that the cells weren't fixed and po= pped. My question is can I fix the slides and then leave them in 4 de= grees fridge either in the alcohol or take them out in a slide box. I= can see not reconsituting them in the saline until ready to stain, but I t= hink I need to fix them and not just leave the fluid sitting in the fridge?= Ahh help!!! Thanks guys and gals =) Sarah Goebel, B.A., HT (ASCP) Histotechnician XBiotec= h USA Inc. 8201 East Riverside Dr. B= ldg 4 Suite 100 Austin, Texas = 78744 (512)386-5107 ___ 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] Vector ABC-HRP
Hi All, This may seem like a no-brainer, but can I dilute Vector's ABC-HRP solution in TBS instead of PBS? I have always used PBS, but we are looking to avoid PBS entirely for one of my projects. Had anyone used TBS to dilute ABC-HRP? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] (no subject)
Good morning, I am trying to make some nice FFPE cell pellet blocks, but I seem to lose a lot of cells along the way (especially when trying to take the cells out of the tube). We are fixing the cells in NBF, spinning them down, adding 70% and spinning down again. At that point, I am trying to scoop out the cells (with a weighing scoop) and wrap them in lens paper for processing. I am losing a lot of the cells during this step, because the pellet is not quite solid. Do you think it would be OK to let the cells air-dry a bit and then take them out for processing? I know this goes against everything I was taught in histology, but I am really at a loss here. Anyone have any hot tips for me? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] (no subject)
Thanks for the tips everyone! I am going to order some agar (histogel) and use it! We are all very excited around here about this, we have been pulling our hair out over these darn pellets. Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Cormier, Kathleen kathleen.corm...@crl.com To: Kim Merriam kmerriam2...@yahoo.com Sent: Wed, May 12, 2010 10:40:19 AM Subject: RE: [Histonet] (no subject) Hi Kim, Have you used Histogel? I have used it in the past (other places of employment) and on samples like yours. I just bought the gel, (not the whole unit, just the gel) scooped some out of the tube, placed into a weight boat, microwaves for a few seconds until liquid, then took a transfer pipet and dropped onto the cell pellet. I then placed the tube/pellet on a cold plate or fridge and wait until solid. I then popped out the now solid pellet, and processed as usual (no baggie/paper) in a cassette. Works like a charm Kathy Kathy Cormier Histology Manager Charles River Laboratories 251 Ballardvale Street Wilmington, MA 01887 Ph: 781-222-6803 Fax: 978-988-8793 kathleen.corm...@crl.com The Charles River 24th Annual Short Course on Laboratory Animal Science will be held June 21-24, 2010, in Newton, MA. Click Here to get more information on this event. Accelerating Drug Development. Exactly. Notice - This email and any files transmitted with it are confidential and may contain privileged and/or proprietary information. You must not disclose this message to another party without Charles River's express written consent. If you are not the intended recipient you must not copy, distribute or use this email or the information contained in it for any purpose other than to notify us. If you have received this message in error, please notify Charles River immediately, and delete it from your system. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Kim Merriam Sent: Wednesday, May 12, 2010 9:24 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] (no subject) Good morning, I am trying to make some nice FFPE cell pellet blocks, but I seem to lose a lot of cells along the way (especially when trying to take the cells out of the tube). We are fixing the cells in NBF, spinning them down, adding 70% and spinning down again. At that point, I am trying to scoop out the cells (with a weighing scoop) and wrap them in lens paper for processing. I am losing a lot of the cells during this step, because the pellet is not quite solid. Do you think it would be OK to let the cells air-dry a bit and then take them out for processing? I know this goes against everything I was taught in histology, but I am really at a loss here. Anyone have any hot tips for me? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet /PREHRfont size=2 face=ArialThis email, its contents and attachments are confidential and may be covered by legal privilege. This email contains information intended only for the person(s) and/or entity named above. The views and opinions expressed are those of the sender and not necessarily those of Charles River Laboratories or its affiliates. Any other distribution, copying, review, use or disclosure is strictly prohibited. If you are not the intended recipient, please delete this message and any attachments without making a copy and advise the sender by return email - thank you. BR/BRThis email and any attachments have been scanned for viruses prior to leaving the Charles River Laboratories network. Charles River Laboratories will not be liable for direct, special, indirect or consequential damages arising from alteration of the contents of this message by a third party or as a result of any virus being passed on. For more information on the range of services that we offer, please visit our website at BA href=http://www.criver.comhttp://www.criver.com/A/B/fontPRE ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC and IF
Hi Anil, Perhaps you are having mouse-on-mouse issues with your IHC (DAB) and you are seeing antibody cross-reactivity with mouse IgGs (you don't mention your IHC detection method, so I could be wrong). You are probably not seeing this cross-reactivity with the IF method, because the labeling is direct. If that is the case, I would be more likely to trust what I saw with the directly-labeled antibody. If you have labeled the monoclonal antibody with FITC and you want to see it with a chromagen, you could use an anti-FITC secondary (Invitrogen makes a nice Rabbit anti-FITC) which you could then detect with an anti-Rabbit polymer (or whatever you normally use to detect a rabbit primary). Good luck, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Thotakura, Anil Kumar a.thotak...@imperial.ac.uk To: Histonet@lists.utsouthwestern.edu Histonet@lists.utsouthwestern.edu Sent: Thu, April 15, 2010 7:14:26 AM Subject: [Histonet] IHC and IF Dear All, I have kind of funny problem. I am doing IHC (substrate reaction suing Dabs reagent) and IF ( direct labeling of my monoclonal antibody with flurochrome). I am working on mouse liver frozen sections. When I did IHC the staining looks like cytoplasm and if I do IF for the same protein I saw nuclear staining. I am unable to conclude whether it is cytoplasm or nuclei staining. Please advice. The monoclonal antibody I used is not commercially available. We made it. Thanks In advance. Many Thanks, Anil Kumar. ___ 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: FW: [Histonet] studying for ASCP certification exam
The NSH sells a set of study guides on histology (maybe 10 of them, on fixation, various special stain categories and the like), I think you will find them quite helpful to see how much you actually remember after reading the textbooks. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Pat Laurie foreig...@gmail.com To: histonet@lists.utsouthwestern.edu Sent: Wed, April 14, 2010 12:02:21 PM Subject: Re: FW: [Histonet] studying for ASCP certification exam I definetly agree with the previous suggestions. Those 3 books are in the top 5 of my collections. Another book that might help you because of the illustrations (special stain color pictures on the test!) is: Theory and Practice of Histological Techniques 5th edition edited by Bancroft and Gamble Unfortunately it is rather pricey. Another good standby because so many books seem to cite it (classic): AFIP Laboratory Methods in Histotechnology latest edition is Prophet et.al. ISBN: 1-881041-00-X Good luck with your test! On Wed, Apr 14, 2010 at 6:58 AM, Ian Montgomery ian.montgom...@bio.gla.ac.uk wrote: Jennifer, HISTOLOGICAL AND HISTOCHEMICAL METHODS. 4th Ed. Kiernan, J.A. In my opinion this is the best histotechnique text currently available. Amazon UK has it in stock for £34.99 and for a text of this quality it's a bargain. If you are studying for an exam this text gives everything you'll need, but more importantly, it is written in a style that is easily understood. Better still, when you have finished studying, the text can sit on a shelf in your lab and become your standard lab manual. No tie in with the author or publisher but I have 2 copies, one in my office and the other in the lab. Ian. Dr. Ian Montgomery, Histotechnology, I.B.L.S. Support Unit, Thomson Building, University of Glasgow, Glasgow, G12 8QQ. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mike Pence Sent: 14 April 2010 14:18 To: Jennifer Campbell; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] studying for ASCP certification exam Also try: Theory and Practice of Histotechnology by Sheehan and Hrapchak. I have heard this is a VERY hard book to get a hold of these days and if you can it will cost! -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jennifer Campbell Sent: Tuesday, April 13, 2010 6:46 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] studying for ASCP certification exam I am in the process of studying for my HT certification exam and was wondering if anyone had any recommendations on text books or study guides they found to be helpful. I am currently studying Histotechnology: A Self-Instructional Text, by Carson and just recieved the BOR study Guide for Histotechnology. Are there any other sources you would recommend? I have taken a look at the suggested reading list on the ASCP website but, there are quite a few books listed. Thanks in advance, Jennifer Campbell ___ 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 -- Patrick Laurie HT(ASCP)QIHC CellNetix Pathology Laboratories 1124 Columbia Street, Suite 200 Seattle, WA 98104 PH: 206-215-5949 plau...@cellnetix.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] NSH online registration
Anyone else having trouble with the online registration for the meeting in Seattle? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Incubation of Ab more then 1 hour in autostainer
Hi Naira, We routinely do a 2 hour incubation in primary, but we make sure that more antibody is on the slide than would be necessary for most other reagents (500-600 ul). We have not had any issues of drying. If you are concerned about it, just make it 2 antibody steps instead of 1 on the machine. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Margaryan, Naira nmargar...@childrensmemorial.org To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Thu, March 18, 2010 3:38:33 PM Subject: [Histonet] Incubation of Ab more then 1 hour in autostainer Hi Histonetters! I have a stupid question but I Have to ask. Does anyone perform an Incubation of Ab that required more then 1 hour (2-3 hours) in autostainer? Does autostainer keep slides wet or slides sometimes are getting dry? I know that slides should not be dry in any step of IHC. Thank you very much! Naira ___ 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
Fw: [Histonet] frozen sections of cartilage
Hi Guys, I never received a response and I was hoping that someone would have some tips. We are embedding and sectioning rat and mouse knee cartilage and I am having trouble keeping it on the slides. Even when I do my IHC by hand, the sections are still lifting off. Any suggestions? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA - Forwarded Message From: Kim Merriam kmerriam2...@yahoo.com To: Histonet histonet@lists.utsouthwestern.edu Sent: Mon, February 22, 2010 2:37:58 PM Subject: [Histonet] frozen sections of cartilage Hi Everyone, Any tips for keeping frozen sections of cartilage from falling off the slides during IHC staining? We should not have to do HIER, but they still fall of the slides quite easily. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ 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] frozen sections of cartilage
Hi Everyone, Any tips for keeping frozen sections of cartilage from falling off the slides during IHC staining? We should not have to do HIER, but they still fall of the slides quite easily. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: [IHCRG] Hamamatsu Nanozoomer
I would like to thank everyone that responded to my questions about these scanners. These answers have been very valuable to my boss and I and will be taken into account when we decide on which system to purchase! Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: James Watson jwat...@gnf.org To: Kim Merriam kmerriam2...@yahoo.com Cc: joel.duckwo...@olympus.com joel.duckwo...@olympus.com Sent: Thu, January 28, 2010 9:15:52 AM Subject: RE: [IHCRG] Hamamatsu Nanozoomer Kim, I use the nanozoomer a lot, we have scanned about 45,000 images in 3 years. I bought it because it does both fluorescence and transmitted light very well. The aperio scanner is good and the software can view both types of scans. The Nanzoomer software has improved greatly in the past 3 years thanks to Olympus and the service has been excellent. I will send you some images and more information when I get to work. I have CC’ed my sales rep, he will make sure someone gets in touch with you today. The cost is about the same as a single instrument from Aperio (unless aperio changed their pricing). Jamie From:Kim Merriam [mailto:kmerriam2...@yahoo.com] Sent: Thursday, January 28, 2010 4:44 AM To: James Watson Subject: Re: [IHCRG] Hamamatsu Nanozoomer Thanks Jamie. When I was at Novartis/NIBRI, I used the Aperio quite a bit (I am at Amgen now, but I remember meeting you a while back at one of the NSH meetings). Anyway, the other Amgen sites use the Aperio, but we are intrigued by the possibility of buying one machine that can do light and IF imaging, also the Z-stack feature is a nice ad-on (although I am not sure how much we would really use it). Based on the information I have gathered, I would be able to pull the images taken from the Nanozoomer and put them on our Aperio server for cross-site conferences. We were actually ready to purchase the Aperio next week, but then my boss found out about the Nanozoomer, which seems to be a better instrument, but I have no idea how much it costs, I cant get anyone from Hamamatsu or Olympus to call me back. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From:James Watson jwat...@gnf.org To: kmerriam2...@yahoo.com kmerriam2...@yahoo.com Sent: Thu, January 28, 2010 1:08:16 AM Subject: FW: [IHCRG] Hamamatsu Nanozoomer Kim, Contact me, I have a Nanozoomer and also know a lot about the Aperio since their headquarters is near here and I have been to their office many times and they have been here. Jamie James Watson HT ASCP Facilities Manager of Histology GNF Genomics Institute of the Novartis Research Foundation Tel 858-332-4647 Fax 858-812-1915 jwat...@gnf.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Liz Chlipala Sent: Wednesday, January 27, 2010 9:09 AM To: kmerriam2...@yahoo.com; Histonet; ih...@googlegroups.com Subject: [Histonet] RE: [IHCRG] Hamamatsu Nanozoomer Kim We have an Aperio Scanner. I'm not quite sure what you are asking but this is what I think from your e-mail. Aperio scans create .svs files these files are considered a modified tiff file. When aperio scans the file is placed into the spectrum database - from there you can create case or project files, etc. These images can be viewed via imagescope. There is no issue with placing other images that have not been generated on the scanscope into the spectrum database, you can upload any image into the spectrum database we have done this in the past. These images can also be viewed with imagescope we have only uploaded single magnification tiff files into the aperio spectrum database. The Hamamatsu Nanozoomer I believe also comes with a database and some viewing sofware. Are you asking if you can view the HN images in the spectrum database? To be honest I'm not sure if you can view the images completed with image scope I would ask your sales rep. I'm sure you are aware that both of these scanners essentially create virtual slides. Not just an image of a slide at one magnification. The viewing software allows you to view the images at different magnifications. I'm not that familar with Visiomorph its the Olympus product and doesn't Olympus sell the HN scanner? I would also think that that the database that comes with the HN scanner should be able to accept other images and should have some conferencing capabilities, I know that the aperio does. I'm not sure if I answered your questions, but I would ask the sales reps from both aperio and Olympus about compatability. With repects to analysis I think that the Visiomorph software will be able to analyze a image generated from the scanscope. If you are working with an HN scanner and the visiomorph software I'm assuming that you will be able to perform batch analysis on selected images from the olympus database. You
[Histonet] Fw: [IHCRG] Dianova rat anti-mouse CD31
Hi - someone emailed me about my methodology, but I cant find the email, so here it is: I tried it @ 1:10, 1:25 and 1:50. All seem viable dilutions for the FFPE xenografts. I used BIocare DIVA (EDTA) HIER in decloaking chamber, primary ab for 2 hours @ RT, Biocare Rat-on-Mouse HRP polymer kit (15 minutes each) + DAB. The path labs (for my company) that are in California and Seattle used the same retreival but different detection methods and got similar results. Kim From:ih...@googlegroups.com [mailto:ih...@googlegroups.com] On Behalf Of Kim Merriam Sent: Wednesday, January 27, 2010 9:23 AM To: Histonet; ih...@googlegroups.com Subject: [IHCRG] Dianova rat anti-mouse CD31 Hi All, I know there was some buzz a while back about this antibody. I have tested it on mouse FFPE xenografts, and I must say, it looks very promising. I compared 2 pieces of the same mouse xenograft; one half of the tumor was fixed in IHC-Zinc (ZInc-Tris) and stained with the BD rat anti-mouse CD31 and the other half of the tumor was fixed in NBF. The NBF-fixed tumor actually looks better! A couple of other folks that are working at the west coast sites of my company have had similar results. We are all very encouraged. I still need to do a bit more testing with some other tissues (and perhaps some additional titrations), but I thought I would pass this along to everyone. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Hamamatsu Nanozoomer
Hi Everyone, I was wondering if any of you folks had experience with the Hamamatsu Nanozoomer slide scanner. We recently learned about it and are considering getting one instead of an Aperio slide scanner. We plan to use Visiomorph image analysis software, so all we really need is the scanner and not all of the fancy software that these companies sell to go with their instruments. The other labs at my company have Aperio systems and the images from the Nanozoomer would need to compatible or made to be compatabile for this to be a viable option for us (for cross-site slide conferencing purposes). We are interested in it because this instrument can be adapted for fluorescence (with Aperio, you need to purchase 2 separate instruments; one for light microscope and one for fluorescence). Any insights would be greatly appreciated. Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Dianova rat anti-mouse CD31
Hi All, I know there was some buzz a while back about this antibody. I have tested it on mouse FFPE xenografts, and I must say, it looks very promising. I compared 2 pieces of the same mouse xenograft; one half of the tumor was fixed in IHC-Zinc (ZInc-Tris) and stained with the BD rat anti-mouse CD31 and the other half of the tumor was fixed in NBF. The NBF-fixed tumor actually looks better! A couple of other folks that are working at the west coast sites of my company have had similar results. We are all very encouraged. I still need to do a bit more testing with some other tissues (and perhaps some additional titrations), but I thought I would pass this along to everyone. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] fumehood
Surgipath makes a nice tabletop fume hood, we have one in our lab and it works quite well. http://www.surgipath.com/us-en/Products/equipment-accessories/fume-hood Good luck! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: rmweber...@comcast.net rmweber...@comcast.net To: histonet@lists.utsouthwestern.edu Sent: Mon, January 18, 2010 12:11:23 PM Subject: [Histonet] fumehood Hi, Does anyone know a good tabletop fumehood for grossing with formalin? I was thinking of one with 60f/sec to 110f/sec. Does that meet the regulations? Marilynn Weber H.T.(ASCP)QIHC ___ 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] requirements for certification
Hi Jen, I am HT certified and have spent my entire career working in research/veterinary pathology. I received my HT years ago when I was working for a preclinical contract lab, I had one of our board-certified veterinary pathologists sign the form for me (I work for a biotech company now and did the same thing when I took my QIHC a couple of years ago). It looks like you have the proper education and experience, I think all you would need is for a board-certified pathologist (either DVM or MD) to sign the form that they are familiar with your work and you should be good to go. Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Jennifer Anderson jander...@halozyme.com To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Wed, January 13, 2010 4:50:53 PM Subject: [Histonet] requirements for certification Hello. I'm thinking about getting my HTL certification. I've been a bench scientist in both academia and biotech for 21 years, which has included pre-clinical animal research and subsequent histological applications. I've set up an histology lab and have a lot of IHC but only basic staining (mostly HE), as well as loads of animal tissue processing and some human tissues. I have a liberal arts degree (an AB) with a biology major and a chemistry minor. We have both a DVM and a few MD's on site, but for pre-clinical and clinical research consult. I would appreciate any insight on getting certified through an online program, how many hours is required, and what kind of mentorship is necessary. Thank you for your insights and time! Jennifer M. Anderson, Scientist Halozyme Therapeutics, Inc. 11388 Sorrento Valley Road San Diego, CA 92121 858-704-8333 jander...@halozyme.commailto: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
[Histonet] opinion please
Help! My refrigerator died over the weekend. This fridge had most of my antibodies and IHC reagents. When I came in, the temperature inside the fridge was a balmy 37C! Do you think any of my reagents are still usable? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] CD68 on FFPE human tissue
What antibodies are people using for this marker? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Floaters in Waterbath
I have always used pieces of phone book paper. Just ask everyone to bring in their old phone books, and tear the papers off at the seam. They provide just the right amount of absorption, are the perfect size, are free and a useful way to recycle! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Angela Bitting akbitt...@geisinger.edu To: Jackie M O'Connor Jackie.O'con...@abbott.com; Stella Mireles estellamire...@gmail.com; histonet-boun...@lists.utsouthwestern.edu Cc: Histonet@lists.utsouthwestern.edu Sent: Fri, October 23, 2009 10:40:58 AM Subject: Re: [Histonet] Floaters in Waterbath We currently have a Quality Improvement Plan in effect to address this issue. Jackie is right about keeping those forcep wells clean. Although we don't swipe Kimwipes over our waterbath after each block, we do it very regularly. Another thing to consider is how often you clean your embedding molds. ~Angie Angela Bitting, HT(ASCP) Technical Specialist, Histology Geisinger Medical Center 100 N Academy Ave. MC 23-00 Danville, PA 17822 phone 570-214-9634 fax 570-271-5916 ! Jackie M O'Connor Jackie.O'con...@abbott.com 10/23/2009 10:23 AM Kim wipes seem to pick up more debris than paper towels, and they pick up much less water. We routinely sweep the waterbath with a kimwipe after each block. You can also pick up floaters from embedding if the forceps are not cleaned between each block. Most embedding centers have multiple wells for forceps - how often do you clean those wells? You'd be amazed at how much gunk accumulates in there! From: Stella Mireles estellamire...@gmail.com To: Histonet@lists.utsouthwestern.edu Date: 10/23/2009 09:11 AM Subject: [Histonet] Floaters in Waterbath Sent by: histonet-boun...@lists.utsouthwestern.edu I know we have all had some problems with floaters in our waterbath at some point in our microtomy career. Our doctors are very picky and I need some tips on keeping an immaculate clean waterbath, but not sacrificing the speed in a regular routine lab. We use the pyrex waterbath and paper towels for wiping our area. Thanks ___ 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 IMPORTANT WARNING: The information in this message (and the documents attached to it, if any) is confidential and may be legally privileged. It is intended solely for the addressee. Access to this message by anyone else is unauthorized. If you are not the intended recipient, any disclosure, copying, distribution or any action taken, or omitted to be taken, in reliance on it is prohibited and may be unlawful. If you have received this message in error, please delete all electronic copies of this message (and the documents attached to it, if any), destroy any hard copies you may have created and notify me immediately by replying to this email. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] harvest DRG from dogs
Hi Everyone, This is slightly off-topic, but does anyone know of a textbook or website that will give information on how to harvest DRG (dorsal root ganglion) from dogs? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC on plasma cells
Hi everyone, I have been asked to do IHC on plasma cells of FFPE human tissue. Anyone have a good antibody (and method) for this procedure? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] histology for kids
Hello All, My company is hosting an in-house science awareness day for local grade-school students. I would love to teach them about histology, but all of the demonstrations need to be done in our conference room (thus, nothing hazardous). Does anyone know of any house-hold dyes (grape juice, food coloring, beet juice, etc) that would stain tissue elements on slides? I would like to bring down some deparaffinized tissues and stain them with something and throw a coverslip on (water-mounted) so that they can look at the tissue with a microscope. I will also bring some already prepared slides (wtih real stains) for them to look at. Any ideas? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] histology for kids
Thanks to everyone that emailed me, I received so many ideas! I will let you all know what I end up donig. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Kim Merriam kmerriam2...@yahoo.com To: Histonet histonet@lists.utsouthwestern.edu Sent: Wednesday, July 22, 2009 9:57:53 AM Subject: [Histonet] histology for kids Hello All, My company is hosting an in-house science awareness day for local grade-school students. I would love to teach them about histology, but all of the demonstrations need to be done in our conference room (thus, nothing hazardous). Does anyone know of any house-hold dyes (grape juice, food coloring, beet juice, etc) that would stain tissue elements on slides? I would like to bring down some deparaffinized tissues and stain them with something and throw a coverslip on (water-mounted) so that they can look at the tissue with a microscope. I will also bring some already prepared slides (wtih real stains) for them to look at. Any ideas? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ 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] anyone tried to get onto the NSH website today?
Good morning everyone, I have tried several times to get onto the NSH website today and I keep getting an error. I am wondering if it is down or if there is something wrong on my end. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Fw: [champ] website for introducing Molecular diagnostics, proteomics etc
Hi All, I subscribe to another listserve and I thought this link might be of interest to people on the histonet (see the link below): Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA - Forwarded Message From: Deborah Payne deborah.pa...@utsouthwestern.edu To: AMP Membership Listserv ch...@lists.asip.org Cc: aacc-proteomics-...@aacclists.org Sent: Friday, June 26, 2009 3:59:58 PM Subject: [champ] website for introducing Molecular diagnostics, proteomics etc Dear Colleagues, The link below is from the NCI and it has several ppt on numerous topics of interest. http://www.cancer.gov/cancertopics/understandingcancer/moleculardiagnostics I thought I would share this. The ppt are a bit simple but still very good. debs * --You are currently subscribed to the CHAMP Listserv. This listserv is exclusively for the membership of the Association for Molecular Pathology. While discussion of commercial technologies and products frequently appears on CHAMP, the listserv is not intended as a medium for distribution of commercial advertisements. We encourage our members to keep this in mind when making postings. AMP will not publish and/or post material that is primarily commercial in nature. Having trouble posting CHAMP messages? Maybe your e-mail address has changed and we need to change your CHAMP listing. Please send us e-mail at: a...@asip.org To unsubscribe, please email Ann Marie Bocus, Membership Manager, at ambo...@amp.org American Society for Investigative Pathology 9650 Rockville Pike Bethesda, MD 20814 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] aggrecan IHC on articular cartilage
Hi Everyone, I was wondering if anyone has done aggrecan IHC on articular cartilage from FFPE/decaled rodent knees. The references and antibodies all call for a funky pretreatment step rather than a routine heat or enzyme (granted all of the references I have found are all for westerns and not IHC). Anyway, the pretreatment calls for incubation with chondroitinase or a combination of chondriotinase/keratinase 1 and 2 (it seems as though the aggrecan is bound up in these other proteins and they need to be digested away to access the aggrecan). Has anyone does this stain on tissues using a more routine retrieval? Also, any antibodies to recommend? Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] HRP-labeled primary antibodies
Hi All, Has anyone had experience doing IHC on FFPE tissues with HRP-labeled primary antibodies? I was wondering what the best way to detect them would be. I assume that going strait to DAB would not work, since no amplification is there. I was thinking of using a biotinyl tyramide step to amplify the signal. Also, do you think the final antibody concentration would need to be higher than with traditional, unlabeled primaries? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] HRP-labeled primary antibodies
I got some great ideas. Thanks everyone! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: anh2...@med.cornell.edu anh2...@med.cornell.edu To: Kim Merriam kmerriam2...@yahoo.com; Histonet histonet@lists.utsouthwestern.edu; ih...@googlegroups.com Sent: Thursday, May 21, 2009 10:48:15 AM Subject: Re: [Histonet] HRP-labeled primary antibodies If there is enough of the antigen in the tissue or sample you can detect without amplification. HRP/DAB is an enzymatic reaction so it is also is amplification. I would try it the straight up way before diving into more complex protocols. Alternatively another option would be that you could try to come in with a secondary antibody (either HRP labeled itself or biotinylated). If your secondary is a polyclonal - which most are - it should still be able to detect the primary even with the HRP attached. Worth trying anyway. -Original Message- From: Kim Merriam kmerriam2...@yahoo.com Date: Thu, 21 May 2009 05:41:44 To: Histonethistonet@lists.utsouthwestern.edu; ih...@googlegroups.com Subject: [Histonet] HRP-labeled primary antibodies Hi All, Has anyone had experience doing IHC on FFPE tissues with HRP-labeled primary antibodies? I was wondering what the best way to detect them would be. I assume that going strait to DAB would not work, since no amplification is there. I was thinking of using a biotinyl tyramide step to amplify the signal. Also, do you think the final antibody concentration would need to be higher than with traditional, unlabeled primaries? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ 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] Floor materials
I know they are expensive, but I think that epoxy floors are best in any kind of lab that uses solvents. Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: louise renton louise.ren...@gmail.com To: Histonet@lists.utsouthwestern.edu Sent: Wednesday, April 29, 2009 3:10:05 AM Subject: Re: [Histonet] Floor materials Vinyl floors can be almost lethally slippery if wax gets on them its ok if staff wear flat shoes but woe betide someone coming in with heels! On 4/28/09, Feher, Stephen sfe...@cmc-nh.org wrote: We're building a pathology lab and we're at project phase where flooring materials is being discussed. The initial choice of the architect is to use sheet vinyl flooring in the working areas of the lab (with the exception of the morgue). I'm not particularly impressed with the way vinyl flooring stands up to stains, solvents and wax. Do any of you have suggestions or experience in a type of flooring for the path lab that is superior to commercial vinyl? Thanks, Steve Stephen A. Feher, MS, SCT (ASCP) Pathology Supervisor Catholic Medical Center 100 McGregor Street Manchester, NH 03102 603-663-6707 sfe...@cmc-nh.org mailto:sfe...@cmc-nh.org ___ 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 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
[Histonet] firefly luciferase
Hi, Is anyone doing firefly luciferase staining in FFPE mouse tissues? Any antibody recommendations? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC for adenoviral vectors
Hi All, I was wondering if anyone has ever done IHC to look for the presence of adenoviral vectors in tissue (ie - to track where the viral vector has spread in the body). Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] HT training
Hi Jennifer, I am HT and QIHC certified and I received both certifications without working in a clinical lab (I did work in a hospital for about 3 months very early on in my career, but it was long before I took the HT exam). I have been in the field for 22 years now, working in biotech, preclinical contract lab and big-pharma. Anyway, you should be able to qualify to take the exam if you work with a boarded DVM pathologist (DACVP), that is how I qualified to take both of them. Good luck! Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA --- On Wed, 2/11/09, Jennifer Anderson jander...@halozyme.com wrote: From: Jennifer Anderson jander...@halozyme.com Subject: [Histonet] HT training To: Histonet@lists.utsouthwestern.edu Date: Wednesday, February 11, 2009, 4:05 PM Hello. I am enjoying this discussion on the pro's and con's and plusses and minuses of certification. I did not know that clinical labs allowed uncertified techs to process human clinical samples - that seems like it would be a huge liability issue. It shouldn't be that way - isn't everyone else in a hospital setting certified somehow? (nurses, radiology...) I am not certified, and I am in a biotech setting (pre-clinical RD). I've just started this position and I'm working with an HT certified person in the lab. We both can trim and gross and cut and process and stain, and troubleshoot. However, she's a professional HT and it shows. She has a lot of clinical background. She has an amazing wealth and breadth of knowledge and skill, and knows what to look for during quality control issues. She doesn't have to take time to peruse the internet or books to get an answer to a histology problem. However if you asked her to do an ELISA or a Western Blot she would probably need some help, unlike myself. I do hope to gain histology knowledge from her, although it's proving to be difficult. I am very interested in developing my skills and learning more about pathology and the science of staining. I would love to be HT certified, but the HT here said I would need to train in a clinical setting for a year, under an ASCP pathologist, which is not likely with the job that I have and being a mom of two. Would anyone know of a less rigorous training program? Something online? Thanks a lot. Jennifer Anderson 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. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Pamela Marcum Sent: Wednesday, February 11, 2009 10:16 AM To: 'Martin, Gary'; Histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] uncertified techs in Histology I am sorry I seemed to have expanded this discussion. I want to be clear on part of the record. I was OJT trained in the 60's. We had even fewer schools and options then. The person who trained me had been trained by the pathologist and the Ann Preece book in histology. She knew what the pathologists we worked with wanted and saw to it that was what they got everyday. When I worked in other places later and continued my education I did learn more about the chemistry and why it worked or failed. I was in research when I took my HT and was told if I used animal tissue I would fail as no one on the board back then was experienced with it. I did not know if it was true so I quickly found a hospital where I could complete everything on human tissue I processed and stained. The person running that lab required me (thank goodness) to process every piece of tissue and do every stain manually. We did not have automated stainers back then so I learned every step. So for those who think I am picking on them for OJT training it is not that I disapprove. I believe histology is too important not to be considered professional field that requires consistent training and education. Many of us old timers have fought hard for the education clause so we would have people who were licensed and fully trained. I did get my BS and more education so I did get more on my own. Pamela A Marcum University of Pennsylvania School of Veterinary Medicine Comparative Orthopedic Laboratory (CORL) 382 W Street Rd Kennett Square PA 19438 610-925-6278 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Martin, Gary Sent: Wednesday, February 11
[Histonet] PhosStop and Complete and cryosections
Hi Everyone, Happy Friday! I am looking to use PhosStop and Complete (both from Roche) in my fixative for cryosections (and cytospins). I have used in in the past in formalin for FFPE xenografts, and now I want to use it as a cryo-section fixative. What I want to know is: has anyone tried this? is this solution re-usable (for more than one rack of slides); will I need a fresh batch for each rack or can I re-use it for several racks during the same day. They are both supposed to be stable, once dissolved, at 4C for 1-month. I don't know how much depletion or inactivation will occur if I use this reagent repeated during the day (it would probably only be about 8 racks of slides in the fixative) should I keep it cold (ie - fix the slides @ 4C) or can I use it at Room Temp (fix the slides at RT) Any insight would be great! Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Complement C3 for mouse FFPE
Hi, Can anyone recommend a good antibody for complement C3 on mouse FFPE tissue? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] training materials
The AFIP book (Laboratory Methods in Histotechnology, edited by Edna Prophet, et al) has nice chapters on specimen orientation and embedding It is not really a training manual, but it has some nice pictures as to how certain tissues should be placed into the molds. Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Daniel Schneider dlschnei...@gmail.com To: histonet@lists.utsouthwestern.edu Sent: Thursday, December 11, 2008 12:20:40 PM Subject: Re: [Histonet] training materials I would be very interested in these suggestions as well, as we would like to improve the quality of skin embedding. Thanks! On Thu, Dec 11, 2008 at 10:59 AM, Jennifer Johnson jmjohnso...@hotmail.comwrote: Can anyone suggest a really good book, atlas, etc. for embedding? The girl that took my place at my last job is having a really hard time (especially with skin) and I told her I would ask the experts. Thanks, Jennifer Johnson, HTL (ASCP) _ Send e-mail faster without improving your typing skills. http://windowslive.com/Explore/hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_speed_122008___ 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] Primary works with ABC but not IF
Hi Nicole, I don't know what kind of secondary antibody you are using, but we have had very good luck with the Alexa-Fluor secondaries. Often, when you go from an amplified chromagenic method (such as ABC) to a less-amplified staining method (such as with a fluorescently-labeled secondary), the primary antibody needs to be retitrated. Perhaps there is not enough amplification with a directly labeled seconday; you may need to add another lay or do additional amplification, such as with tyramide - we are having very good luck with Invitrogen's TSA kits that contain AlexaFluor dyes. The Perkin Elmer FITC-TSA kits also work well, but cost about 3X as much as Invitrogens. If you send more information about your staining protocols, we could probably give you more specific advice. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Patten, Nicole (NIH/NIAAA) [F] [EMAIL PROTECTED] To: histonet@lists.utsouthwestern.edu Sent: Wednesday, December 10, 2008 10:09:45 AM Subject: [Histonet] Primary works with ABC but not IF Hello- I am in a situation where my primary antibody works using the ABC method, but I do not see staining with immunofluorescence. My IF protocol works well with other antibodies so I do not know why it's not working with this particular antibody. Does anybody have any suggestions? I have considered using an avidin-Alexa Fluor conjugate but I am not sure if this is even possible (I am new to IHC) or how I would go about it. Would I need to first biotinylate my primary? Does anyone have any advice/protocols? Thanks! Any help would be greatly appreciated. Nicole Patten Post-Baccalaureate Fellow/IRTA NIAAA/National Institutes of Health ___ 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 on fresh frozen
We use acetone/ethanol (as recommended by Gayle Callis in one of her NSH classes). The slides look great; nice crisp nuclei (which you just won't get with acetone alone). We air dry the slides and fix for 10 minutes in RT acetone/ethanol (75% acetone/25% absolute ethanol) and then put the slides into wash buffer and continue with IHC. I have heard that this fixative could be problematic with some antigens, but we have not found that to be the case with anything we have used in my lab. Insulin should be fine. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Patti Loykasek [EMAIL PROTECTED] To: FU,DONGTAO [EMAIL PROTECTED]; histonet@lists.utsouthwestern.edu Sent: Wednesday, December 3, 2008 11:38:07 AM Subject: Re: [Histonet] IHC on fresh frozen After hearing a presentation by Sharon Lear describing some low temp antigen retrieval that she did, we changed our method for frozen sections. We fix the frozen sections in 10% nuetral buffered formalin for 30'-60', rinse, then do a gentle pretreatment. The gentle pretreatment is usually 10mM citrate buffer pH6 at 70 degrees C for 30'. Slides are cooled, and usual IHC done. This has worked well for us. Our staining with this method is more reliable intense than with previous methods. Patti Loykasek Hi, all Recently I did some IHC(chromagen methods) on mouse fresh frozen tissues, mainly using insulin antibody on pancreas. The image is much fuzzier compare to paraffin embedding tissue. And the staining also smeared to acinar cells which surround the islet. I airdried slide(30min) and used a general acetone method(-20C 5min) to fix the tissue before I did IHC. How can I get a relatively sharp staining on the fresh frozen tissue?Does anyone here have any experience on it? Any suggestions? Many thanks and have a nice day, Ann ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This e-mail message, including any attachments, is for the sole use of the intended recipients and may contain privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by e-mail and destroy all copies of the original message, or you may call PhenoPath Laboratories, Seattle, WA U.S.A. at (206) 374-9000. ___ 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] anti-mouse ferritin
Hi, Any recommedations for anti-mouse ferritin antibodies (looking to stain FFPE mouse tissue). Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: [IHCRG] HIER
In my experience, you can use the Biocare Decloaker with their default program for most protocols (I believe it heats @ 125C for 10 seconds and then 90C for 10 minutes, but dont quote me on that because I am not sure). I do not preheat my buffer when I use the pressure cooker, since it heats up pretty quickly. I have used a lower-temp protocol for some phospho-proteins (90C for 30 minutes), but that is the exception and not the rule. I also use a Black Decker steamer on a regular basis. I always preheat the buffer (in the microwave) before putting it in the steamer, because the steamer heats slowly and does not get to temps that are as high as the pressure cooker (I check the steamer regularly and it gets to about 100C or so on most days). I usually steam the slides for 45-60 minutes. In the case of both heating systems, I usually let the slides cool for about 10 minutes and then rinse them in running water. I have heard of other people cooling their slides for up to an hour and still others that do not cool at all. I am not sure that how long you cool the slides really has an impact on anything (IMHO, it is all about the heating and not the cooling). As far as pH is concerned, that a whole other topic. For most antibodies, a routine citrate or EDTA buffer works fine (pH 6-8 or so), but I have used the high-pH buffers on occasion (usually for nuclear proteins). I work in a biotech/ discovery research lab, so my particular lab does not need to follow strick regulatory guidelines (although I try to run the lab in a GLP-like fashion). Just my 2-cents. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Perry, Margaret [EMAIL PROTECTED] To: [EMAIL PROTECTED] [EMAIL PROTECTED]; histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Wednesday, October 22, 2008 12:28:24 PM Subject: [IHCRG] HIER We have been using the microwave HIER and have had good results, however when our microwave bits the dust I would like to have a pressure cooker method in place. I feel the pressure cooker is more consistent for all the slides. We are a veterinary diagnostic lab and I would like to have some idea of where to begin. I have looked at different protocols and they often indicate HIER in a pressure cooker but do not give the details. I currently use citrate buffer pH 6. I put the slides in refrigerated buffer and microwave on high for 1 min 45 sec. or until the buffer just starts to boil. I then set the microwave on 10% power for 10 minutes. Afterward the slides are allowed to cool in the microwave for 1 hour. We have a biocare Decloaker Chamber and I would appreciate help with the program I should use. Do you start with cold buffer or should I prewarm it? What temperature should I use? How long should I maintain the temperature? How long should it be before I remove the slides? I also am working with a new protocol that calls for heating in a steamer. Should the temperature of the buffer be warm, cold or room temp when I start? Thank you. Margaret Perry HT (ASCP) IHC Lab Manager Veterinary Science Animal Disease Research and Diagnostic Lab South DakotaState University Box 2175 North Campus Drive BrookingsSD 57007 --~--~-~--~~~---~--~~ You received this message because you are subscribed to the Google Groups ihcrg group. To post to this group, send email to [EMAIL PROTECTED] To unsubscribe from this group, send email to [EMAIL PROTECTED] For more options, visit this group at http://groups.google.com/group/ihcrg?hl=en -~--~~~~--~~--~--~--- ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] conjugated anti-FITC antibody
Hello All! I was wondering if anyone was aware of an anti-FITC antibody that was conjugated to either HRP or Biotin (eg HRP-rabbit anti-FITC or Biotinylated rabbit anti-FITC). If such a thing exists, can you tell me who sells it? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
