[Histonet] MUC3
Hi Histonetters Does anyone use MUC3(mucin) antibody that is working on FFPE human tissue? Many Thanks Marilyn Tyler Medical School UCT Dept of Surgery J50/26 or 30 Surgical research. OMB Groote Schuur Hospital Observatory 021-4066227 ### UNIVERSITY OF CAPE TOWN This e-mail is subject to the UCT ICT policies and e-mail disclaimer published on our website at http://www.uct.ac.za/about/policies/emaildisclaimer/ or obtainable from +27 21 650 9111. This e-mail is intended only for the person(s) to whom it is addressed. If the e-mail has reached you in error, please notify the author. If you are not the intended recipient of the e-mail you may not use, disclose, copy, redirect or print the content. If this e-mail is not related to the business of UCT it is sent by the sender in the sender's individual capacity. ### ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Fixation
I would like to have some informations about fetal brain fixation.. In general, it is in use the fomalin, but during the cut it is possible to see that in the deep layer the formalin doesn't arrive... So, Could you give some suggestion to improve this technique?! Thanks in advance Best Giulia Zunino, PhD Student Laboratory of Molecular Neuropathology Centre for Integrative Biology (CIBIO), University of Trento ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Embedding centers
Good morning, I have a tissue tek embedding center. The hot plate no longer works. I have been told I can not get it replaced until July and it is too expensive to repair. I have looked into barrowing or getting a loaner from someone who has a back-up. That has not worked. Anyone have any ideas on maybe a fix? Thanks Marcy CONFIDENTIALITY NOTICE: This e-mail communication and any attachments may contain confidential and privileged information for use by the designated recipients named above. They are intended solely for these recipients. If you are not the intended recipient, you are hereby notified that you have received this communication in error and that any review, disclosure, dissemination, distribution or copying of it or its contents is prohibited. If you have received this communication in error, please notify Calvert Memorial Hospital immediately by telephone at (410)535-8282 and destroy all copies of this communication and any attachments. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Fixation
The fixation is not complete because fixation time is too short. Please go to http://www.histosearch.com/rene.html where you can find two articles about formalin fixation where this issue is discussed. René J. From: Giulia Zunino giuli.zun...@gmail.com To: histonet@lists.utsouthwestern.edu Sent: Wednesday, March 13, 2013 6:31 AM Subject: [Histonet] Fixation I would like to have some informations about fetal brain fixation.. In general, it is in use the fomalin, but during the cut it is possible to see that in the deep layer the formalin doesn't arrive... So, Could you give some suggestion to improve this technique?! Thanks in advance Best Giulia Zunino, PhD Student Laboratory of Molecular Neuropathology Centre for Integrative Biology (CIBIO), University of Trento ___ 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] RE: Embedding centers
Maybe one of the used equipment vendors would consider a short-term lease with option to buy. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of MARCELLYN A. STONE Sent: Wednesday, March 13, 2013 7:14 AM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] Embedding centers Good morning, I have a tissue tek embedding center. The hot plate no longer works. I have been told I can not get it replaced until July and it is too expensive to repair. I have looked into barrowing or getting a loaner from someone who has a back-up. That has not worked. Anyone have any ideas on maybe a fix? Thanks Marcy CONFIDENTIALITY NOTICE: This e-mail communication and any attachments may contain confidential and privileged information for use by the designated recipients named above. They are intended solely for these recipients. If you are not the intended recipient, you are hereby notified that you have received this communication in error and that any review, disclosure, dissemination, distribution or copying of it or its contents is prohibited. If you have received this communication in error, please notify Calvert Memorial Hospital immediately by telephone at (410)535-8282 and destroy all copies of this communication and any attachments. ___ 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] CD68
Good Morning! CD68 (for the BOND)is backordered until May and I am looking for recommendations - Cellmarque or Biocare? Is there something better? I appreciate your input. Nancy Dubuque, IA NOTICE: This email may contain legally privileged information. The information is for the use of only the intended recipient(s) even if addressed incorrectly. If you are not the intended recipient, please notify the sender that you have received it in error and then delete it along with any attachments. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Fixation
Rene is correct, formalin fixes slowly so more time is needed. Raising the concentration of formaldehyde will not help. Adding a small amount of glutaraldehyde (0.25 to 0.5%) will fix more quickly but may have a negative effect on immunostaining. Geoff On 3/13/2013 6:31 AM, Giulia Zunino wrote: I would like to have some informations about fetal brain fixation.. In general, it is in use the fomalin, but during the cut it is possible to see that in the deep layer the formalin doesn't arrive... So, Could you give some suggestion to improve this technique?! Thanks in advance Best Giulia Zunino, PhD Student Laboratory of Molecular Neuropathology Centre for Integrative Biology (CIBIO), University of Trento ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- -- ** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583; fax: -4029 mcaul...@umdnj.edu ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: CD68
We use Ventana's ready to use for the Ultra, but it is clone KP1. You can get the same clone in a concentrate from Cell Marque. Sue Sue Hunter, Supervisor Advanced Diagnostics Beaumont Health System Royal Oak MI 248-898-5146 shun...@beaumont.edu -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Nancy Schmitt Sent: Wednesday, March 13, 2013 9:52 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] CD68 Good Morning! CD68 (for the BOND)is backordered until May and I am looking for recommendations - Cellmarque or Biocare? Is there something better? I appreciate your input. Nancy Dubuque, IA NOTICE: This email may contain legally privileged information. The information is for the use of only the intended recipient(s) even if addressed incorrectly. If you are not the intended recipient, please notify the sender that you have received it in error and then delete it along with any attachments. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] SMC Immuno and Elastin combo?
Hello, I've read some papers which reference the combination of smooth muscle cell actin immunostaining with a Verhoeff's elastin counterstain. Has anyone ever done this and if so, do you have protocol you'd be willing to share? I'm staining mouse and rat lung and have a SMC protocol working that uses an Abcam antibody and NovaRed chromagen (peroxidase). I've also done EVG staining so I'm familiar with the general process. The combination is what I'm not sure of - I gave it a try for the heck of it and wiped out the immunostain. We've just started looking at this, so nothing is set in stone and I'd be grateful for any advice/ procedural help. Thanks in advance! Patti Bourassa Karos Pharmaceuticals -- *Patti Bourassa Senior Scientist Karos Pharmaceuticals (203) 535-0540, ext 207* ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] SMC Immuno and Elastin combo?
Patti, The immunostaining probably was not wiped out, rather just covered up with the red color of the VG counterstain. You might try a chromogen that expresses in a different color. Or, you might leave off the van gieson part of the EVG (staining only the elastic tissue) and pick a counterstain of another color. Basically, you just need to make sure that none of the stains used have the same color outcome. I have done this combo in the past. Never really finished the project because the issue I had was that the lizard tissue I was working with just would not stay on the slide no matter what I tried. But, I do remember that I treated them as two separate stains. I did the actin staining first, rinsed it well, then started in on the elastin stain. You probably have a protocol for each of these and you just need to combine them. Nancy Thomas Stowers Institute -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Patricia Bourassa Sent: Wednesday, March 13, 2013 10:58 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] SMC Immuno and Elastin combo? Hello, I've read some papers which reference the combination of smooth muscle cell actin immunostaining with a Verhoeff's elastin counterstain. Has anyone ever done this and if so, do you have protocol you'd be willing to share? I'm staining mouse and rat lung and have a SMC protocol working that uses an Abcam antibody and NovaRed chromagen (peroxidase). I've also done EVG staining so I'm familiar with the general process. The combination is what I'm not sure of - I gave it a try for the heck of it and wiped out the immunostain. We've just started looking at this, so nothing is set in stone and I'd be grateful for any advice/ procedural help. Thanks in advance! Patti Bourassa Karos Pharmaceuticals -- *Patti Bourassa Senior Scientist Karos Pharmaceuticals (203) 535-0540, ext 207* ___ 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] SMC Immuno and Elastin combo?
Patti, You're using a mouse monoclonal antibody on mouse tissue, so be aware that the secondary link antibody will bind to any mouse IgG it finds in the tissue (normal plasma cells, IgG in normal sera present inside blood vessels, or as infiltrating interstitial normal sera, etc.), not just to the primary anti-Actin antibody. You may have to use a mouse-on-mouse kit for the Actin staining portion, if you see too much nonspecific background in your tissue. Jan Shivers UMN Vet Diag Lab On Wed, Mar 13, 2013 at 10:58 AM, Patricia Bourassa pboura...@karospharma.com wrote: Hello, I've read some papers which reference the combination of smooth muscle cell actin immunostaining with a Verhoeff's elastin counterstain. Has anyone ever done this and if so, do you have protocol you'd be willing to share? I'm staining mouse and rat lung and have a SMC protocol working that uses an Abcam antibody and NovaRed chromagen (peroxidase). I've also done EVG staining so I'm familiar with the general process. The combination is what I'm not sure of - I gave it a try for the heck of it and wiped out the immunostain. We've just started looking at this, so nothing is set in stone and I'd be grateful for any advice/ procedural help. Thanks in advance! Patti Bourassa Karos Pharmaceuticals -- *Patti Bourassa Senior Scientist Karos Pharmaceuticals (203) 535-0540, ext 207* ___ 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] Issues with Shandon Pathcentre
Hi all, I have some issues with Shandon Pathcentre. Though the machine is kind of old it was very perfect in shape and running good. I am posting this request to see if any of other labs faced this issue before and if any one can give me suggestions. It was running very fine and perfect, until it showed an error recently saying temperature too high on rotary chamber. I checked the settings and it was on 35 which I assume is fine. All my waxes are also at fine temperature. I changed all the waxes and reagents to see if its a false sensor. But did not help. I changed the protocol also from 35 to ambient temperature, did not work. I reset the memory completely , did not work. I tried calling some repair places like Dolbey-Jamison and few other places. Most of them say they dont work with this machine any more. I would appreciate if any one can suggest me any advice, regarding handling the issue differently or if any one know a lab repair service who deals with this kind old machines is much appreciated. Thank you, Lakshmi Kammili, Sr. Research Associate, School of medicine and health sciences, George Washington University, Ross hall, #725 2300 eye St, NW Washington DC. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Placenta - Requests for Homeopathic remedy usage
We indeed discussed placenta encapsulation on Histonet about a year ago. http://lists.utsouthwestern.edu/pipermail/histonet/2012-March/061671.html There are also placental homeopathic remedies: http://lists.utsouthwestern.edu/pipermail/histonet/2012-March/061671.html though I'm not sure that soaking a placenta in brandy for a week is exactly what Dr. Hahnemann prescribed. Bob Richmond Samurai Pathologist Maryville TN ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Alizarin Red S Staining Protocol
Good afternoon, In the past I have been using a 40mM Alizarin Red S Solution from Millipore to stain my cell cultures but I am now seeking an alternative. I plan on preparing fresh Alizarin Red S by adding the powder to 100ml of distilled water. In reviewing the literature, I have come across many labs using 1% ARS or 2% ARS and I am not sure if this concentration significantly matters. Furthermore, I understand some people use McGee-Russell's procedure using a pH of ~4.2 whereas others use Dahl's procedure using a pH of ~6.3. However using a pH of 4.2 seems illogical to me as this could remove some calcium from the monolayer. Currently I plan on fixing the cells with 4% Paraformaldehyde in PBS (pH 7) for 15 minutes, washing twice with water and then staining the cells with 2% ARS in water (pH 6.3). Do you see any problems with this? Also, should I stain for 5 minutes or roughly an hour with the ARS dye? I have had some problems with extracting this dye in the past. The 10% acetic acid does not seem to remove all of the dye and the results are variable. Note: before I extract the cells with acetic acid I wash the cells four times with water. Regards, Sean Tighe ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] All Members Working with Bone, Biomaterials and Medical Device Implants
On May the 4th, 2013, Polysciences, Inc. is hosting a full day educational event focusing on the Histological Applications Techniques for Bone, Biomaterials and Medical Device Implants. On behalf of the speakers (Bob Skinner, Philip Seifert, Valantou Grover and Jack Ratliff) contributing to the educational content for this event, we would like for you to join us in Cambridge, MA at the Hyatt Regency Cambridge (overlooking Boston). With our combined histology experience of 90+ years in working with clinical and preclinical specimens pertaining to bone, biomaterials and medical device implants, you can expect to further your knowledge, understanding and training of specialized histology techniques associated with these specimen types and the applicational relevance of these techniques used in the evaluation safety and efficacy of therapeutic treatments. Registration for this event is now open with Early Bird registration set to end one week from Friday on March 22nd. The National Society for Histotechnology (NSH) will be providing 6 CEUs and Polysciences, Inc. will be providing a discount to any current NSH member as outlined below: WORKSHOP FEES Before March 22, 2013: $149.00 for NSH Members / $199.00 for Non-Members After March 22, 2013: $179.00 for NSH Members / $229.00 for Non-Members For the complete details of this full day Histological Applications Techniques for Bone, Biomaterials and Medical Device Implants event and to register online, please visit the following link: http://www.polysciences.com/Interactive-Histology-Forum-About/185/ and sign up today to participate in the discussion of these specialized histology specimen applications and techniques that are rarely shared or even discussed on Histonet! You will not want to miss out on the information presented by 4 expert speakers in the field, all course materials, meals and a complete program book also containing technique specific protocols that you can repeat back in your lab! We hope to see you in Cambridge (Boston) and May the 4th be with you! Best Regards,Jack Ratliff ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] SMC Immuno and Elastin combo?
Nancy, The red of the classic VG counterstain stains the collagen. Muscle will appear yellow. If the yellow is obscuring the brown DAB then after VG staining wash the slides in water. This will remove the picric acid staining leaving the muscle brown. Since Nova red is red (I assume since I have not had cause to use it - I'm a DAB man!), I would use Picro Indigo carmine or Picro-Light green. Picro-indigo Solution (Ortiz-Hidalgo C Abelardo Gallego(1879-1930)and his contributions to histotechnology: The Gallego stains Acta histochemica1 13(2011):189-193): Picric acid (saturated solution)100ml Indigo carmine 0.25mg Distilled water 15ml Whippy's Elastic Stain (Whippy P A Stain to Try - Whippy's Elastic Stain Histograph Jan 2008:7): Light Green Stock Solution Light Green 2 gm Distilled water 100 ml Glacial Acetic Acid 2 ml Place Light Green and distilled water into a conical flask and mix well. Add Acetic acid and mix. Filter and pour into stock bottle. Light Green - Picrate (Whippy's) Solution Saturated aqueous picric acid 10 ml Light Green stock solution5 drops Pour Picric Acid into a conical flask. Add Light Green solution, stir until dissolved. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Thomas, Nancy Sent: Thursday, 14 March 2013 3:33 AM To: 'Patricia Bourassa'; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] SMC Immuno and Elastin combo? Patti, The immunostaining probably was not wiped out, rather just covered up with the red color of the VG counterstain. You might try a chromogen that expresses in a different color. Or, you might leave off the van gieson part of the EVG (staining only the elastic tissue) and pick a counterstain of another color. Basically, you just need to make sure that none of the stains used have the same color outcome. I have done this combo in the past. Never really finished the project because the issue I had was that the lizard tissue I was working with just would not stay on the slide no matter what I tried. But, I do remember that I treated them as two separate stains. I did the actin staining first, rinsed it well, then started in on the elastin stain. You probably have a protocol for each of these and you just need to combine them. Nancy Thomas Stowers Institute -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Patricia Bourassa Sent: Wednesday, March 13, 2013 10:58 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] SMC Immuno and Elastin combo? Hello, I've read some papers which reference the combination of smooth muscle cell actin immunostaining with a Verhoeff's elastin counterstain. Has anyone ever done this and if so, do you have protocol you'd be willing to share? I'm staining mouse and rat lung and have a SMC protocol working that uses an Abcam antibody and NovaRed chromagen (peroxidase). I've also done EVG staining so I'm familiar with the general process. The combination is what I'm not sure of - I gave it a try for the heck of it and wiped out the immunostain. We've just started looking at this, so nothing is set in stone and I'd be grateful for any advice/ procedural help. Thanks in advance! Patti Bourassa Karos Pharmaceuticals -- *Patti Bourassa Senior Scientist Karos Pharmaceuticals (203) 535-0540, ext 207* ___ 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 * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the
[Histonet] hot copy
http://www.petfriendly.es/llahg/hxlke.wcdarfmxxhbo ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Alizarin Red S Staining Protocol
We always used a 2% Alizarin red S (never tried it with 1%. I'm guessing that you would just have to leave it in longer.) We pH it to 4.1-4.3 with 0.5% ammonium hydroxide. I don't remember what the pH is when we begin, but note, we're adding a BASE to bring the pH up to ~4.2. So alizarin red S must be fairly acidic when dissolved in water. I was told that this is a chelating solution for soft metals. It therefore is not a specific stain for calcium, as it will also stain magnesium, manganese, barium, and strontium. However, these metals are usually not in very high concentrations. But to make it more specific for calcium, a pH of 4.1-4.3 is recommended. I've never done this stain on cell cultures, only on formalin fixed, paraffin embedded, 5 um thick sections. And we find that staining between 1-2 minutes works the best. This is a very sensitive stain. The longer the slide is left in the solution, the more and more the alizarin red S stains the background, to the point that it becomes difficult to tell positive calcium from background staining. Every cell has calcium in it (think membrane transport), and eventually, all the cells are going to pick up background orange color, not just the lesions with calcium. You mention extraction with acetic acid. Extracting what? Are you trying to extract the background staining? I don't know if that will work. This is a chelating agent . . . the CLAW! Once chelating agents hook up with the metal, they don't let go. I just don't happen to know if acid will disrupt this chelating bond. So it would be better to cut the time way down (try 1-2 minutes, see how it looks), and don't have so much background staining to begin with. Let us know how it turns out. Peggy A. Wenk, HTL(ASCP)SLS Beaumont Health System Royal Oak, MI 48073 Opinions expressed are mine, and do not reflect upon my place of employment. -Original Message- From: Tighe,Sean T Sent: Wednesday, March 13, 2013 4:48 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Alizarin Red S Staining Protocol Good afternoon, In the past I have been using a 40mM Alizarin Red S Solution from Millipore to stain my cell cultures but I am now seeking an alternative. I plan on preparing fresh Alizarin Red S by adding the powder to 100ml of distilled water. In reviewing the literature, I have come across many labs using 1% ARS or 2% ARS and I am not sure if this concentration significantly matters. Furthermore, I understand some people use McGee-Russell's procedure using a pH of ~4.2 whereas others use Dahl's procedure using a pH of ~6.3. However using a pH of 4.2 seems illogical to me as this could remove some calcium from the monolayer. Currently I plan on fixing the cells with 4% Paraformaldehyde in PBS (pH 7) for 15 minutes, washing twice with water and then staining the cells with 2% ARS in water (pH 6.3). Do you see any problems with this? Also, should I stain for 5 minutes or roughly an hour with the ARS dye? I have had some problems with extracting this dye in the past. The 10% acetic acid does not seem to remove all of the dye and the results are variable. Note: before I extract the cells with acetic acid I wash the cells four times with water. Regards, Sean Tighe ___ 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
