[Histonet] FW: Ventana vs. Leica
This is how professional histotechs treat each other? A very sad commentary in a country where people are afforded free speech. Maria Katleba MS HT(ASCP) Pathology Dept. Mgr Queen of the Valley Medical Center 1000 Trancas Street Napa CA 94558 (707) 252-4411 x3689 direct (707) 226-4385 pager (707) 294-9229 cell- anytime From: Joe Jones [mailto:joe_j_jone...@yahoo.com] Sent: Tuesday, September 07, 2010 4:28 PM To: Maria Katleba Subject: Ventana vs. Leica Maria, Please stop embarassing those of us who know you and have to work with you. You are clueless about Ventana's business, let alone anyone elses. Your postings on Histonet scream "idiot", so please stop. Ventana paying for a company's waste has nothing to do with a business plan, you dim ass. They pay it after some customer's bring up the additional cost when weighing their options. Fix the "machine" (it's not a machine you stupid tool)? If you knew anything about how instrument's are designed, you'd realize a company doesn't go back and redo an instrument. Now shut up and do us all a favor. Toni brings up a very good point you know it's pretty bad when a company is willing to pay your waste costs... Not a good business plan. Why not 'fix' the machine so that it's more "green" Maria Katleba MS HT(ASCP) Notice from St. Joseph Health System: Please note that the information contained in this message may be privileged and confidential and protected from disclosure. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Silver
I thought Histotechs were supposed to have purple thumbs. :) (You know, gardeners have green thumbs...) Claire From: histonet-boun...@lists.utsouthwestern.edu on behalf of O'Donnell, Bill Sent: Tue 9/7/2010 4:15 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Silver I know... I should wear gloves when doing a GMS.I... know... that. (sorry, I thought this was Facebook for a second) Have a great week! - Sir Bill of the Blackened Thumb ___ 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] Silver
I know... I should wear gloves when doing a GMS.I... know... that. (sorry, I thought this was Facebook for a second) Have a great week! - Sir Bill of the Blackened Thumb ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] caris and genoptix experience
Gee... It's not like WE have work to do or anything. Thanks for the heads up about these companies. Claire From: histonet-boun...@lists.utsouthwestern.edu on behalf of kim.dona...@bhcpns.org Sent: Sat 9/4/2010 10:55 AM To: histot...@imagesbyhopper.com Cc: histonet@lists.utsouthwestern.edu; histonet-boun...@lists.utsouthwestern.edu Subject: RE: [Histonet] caris and genoptix experience I had exactly the same experience with the same company. It's a bad mark right off the bat with me if vendors just walk in the door and expect me to drop what I am doing. Kim Donadio Pathology Supervisor Baptist Hospital 1000 W Moreno St. Pensacola FL 32501 Phone (850) 469-7718 Fax (850) 434-4996 Sent by: histonet-boun...@lists.utsouthwestern.edu 09/03/2010 09:51 PM To "'Tench, Bill'" , cc Subject RE: [Histonet] caris and genoptix experience Very interesting. I recently had a very pushy Genoptix rep at my facility. Wanted to take a LOT of my time, but as I was walking out the door (the rep came unannounced), they only got about 10 minutes. They were quick to point out that wasn't enough time ... well, um ... make an appointment!! -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tench, Bill Sent: Friday, September 03, 2010 4:13 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] caris and genoptix experience We have had almost identical experiences with both organizations. Caris has been informed that it is not welcome; genoptix is on the same list Bill Tench Associate Dir. Laboratory Services Chief, Cytology Services Palomar Medical Center 555 E. Valley Parkway Escondido, California 92025 bill.te...@pph.org Voice: 760- 739-3037 Fax: 760-739-2604 [None] made the following annotations - Confidential E-Mail: This e-mail is intended only for the person or entity to which it is addressed, and may contain information that is privileged, confidential, or otherwise protected from disclosure. Dissemination, distribution, or copying of this e-mail or the information herein by anyone other than the intended recipient is prohibited. If you have received this e-mail in error, please notify the sender by reply e-mail, and destroy the original message and all copies. - ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet No virus found in this incoming message. Checked by AVG - www.avg.com Version: 8.5.441 / Virus Database: 271.1.1/3104 - Release Date: 09/03/10 06:34:00 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet - All electronic data transmissions originating from or sent to Baptist Health Care Corporation (BHC) are subject to monitoring. This message along with any attached data, are the confidential and proprietary communications of BHC and are intended to be received only by the individual or individuals to whom the message has been addressed. If the reader of this message is not the intended recipient, please take notice that any use, copying, printing, forwarding or distribution of this message, in any form, is strictly prohibited and may violate State or Federal Law. If you have received this transmission in error, please delete or destroy all copies of this message. For questions, contact the BHC Privacy Officer at (850) 434-4472. Rev.10/07. ___ 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] trephine protocols
We are experiencing some problems with our trephine bone marrow biopsies. Could anyone share with me the amount of time they are generally using for decalcification and what reagents you are using? We are also experiencing fragmentation in some of the slides. Thanks James Vickroy BS, HT(ASCP) Surgical and Autopsy Pathology Technical Supervisor Memorial Medical Center 217-788-4046 This message (including any attachments) contains confidential information intended for a specific individual and purpose, and is protected by law. If you are not the intended recipient, you should delete this message. Any disclosure, copying, or distribution of this message, or the taking of any action based on it, is strictly prohibited. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Rat tendon processing
Hi, I am processing rat infraspinatus tendons for Masson Trichrome staining. I've had trouble with routine processing protocols and read that adding 4% phenol to the 95% alcohol solutions may help soften the tissue to make it easier to cut. Has anyone ever tried this, or have other suggestions for preventing the tendon from getting so brittle it falls out of the paraffin? Also, should I be extending the time that the tendon spends in each alcohol station or shortening it? I am processing manually since we do not have access to an automatic processor. Thanks, Kristen ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Negative Control Tissue - No / Few Macrophages
Hello, Does anyone know of a good control tissue absent of (or with few) macrophages? I have seen a number of postings regarding tissue positive for various inflammatory markers (F4/80, MOMA. CD68). I am running F4/80 (Serotec, Rat anti-ms F4/80) on ms. tissue. I have a number of positive control tissues -- liver, spleen, etc. Any suggestions would be very helpful. Thank you. Gustave Gustave T. Hebert Research Scientist I Pfizer Cambridge, MA 02140 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Losing Sections While Dehydrating
Hello Everyone! I have searched the archives because I am sure this is a problem that has been encountered before, but I have been unsuccessful in finding the right thread. I ran an IHC series dilution on a new primary antibody for NeuN. I am using 35 um rat brain sections that were cut on a cryostat and stored in Cryoprotectant until ICC began. After finishing the ICC protocol, I mounted the slides that were floating in phosphate buffered saline onto slides that I subbed two weeks ago (.5% gelatin subbing). I allowed the slides to dry from Friday afternoon until Tuesday afternoon. I tried to dehydrate them for coverslipping and when they went into the dH20 bath, some of the sections began to fall off of the slides. This continued in the 50% Alcohol bath. All tissue that made it through those first two baths in the series stayed on throughout, but I did lose quite a few sections, especially those that had the lowest concentration of primary antibody. My experience has been that highly concentrated primaries make the tissue a bit sticky, but the best staining generally comes from the least concentrated. Does anyone know what the problem might be? I think it might be a subbing problem but I'm not sure. Thanks in advance. Amanda Madden Research Assistant P.S. The subbing protocol that I used only called for one "dip" into the gelatin solution, and didn't call for any type of acid wash. Also, our cryostat is a Leica CM3050S and I was wondering if any of your labs have established a working range in terms of humidity. Our lab has been experiencing very high humidity levels (up to almost 80%), so we were wondering if anyone has established that cutting cannot be done unless the humidity is under x%. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Problem with liver fixation
Formalin fixes slowly, even more slowly at 4 degrees C. Even though the fix may penetrate, fixation will not be complete. I suggest a formalin+alcohol+acetic acid fix at room temp for 24 hours. There are many, many variations of this fix. Lillie recommends 85 ml of 95% EtOH, 10 ml conc. formalin (37-40% formaldehyde), 5 ml glacial acetic acid. I agree with Rene, Bouin will not make a difference. Alchoholic Bouin is also worth a try. Geoff Itai Moshe wrote: Dear All, I'm trying to use liver sections in paraffin blocks for IHC, but get no signal at all, doesn't matter what antibody i'm using. I'm using section about 3mm thick (the natural thickness of mouse liver) and about 5-7mm Width and Length. My fixation protocol is like this: 1) immediately after killing the mouse i'm putting the sections in a fixation solution that is made from: 10ml formaldehyde 37%, 5ml PBSx20, 85ml DDW - pH 7, 24Hr at 4C. 2) ETOH 70%, ETOH 80%, ETOH 96%, ETOH 100% x2 - each for 1Hr at RT 3) Xylen x2 - each for 1Hr at RT. 4) Paraffin x3 - each at 60C for 1Hr. Before doing IHC: 1) Xylen x2 - each for 10Min at RT 2) ETOH 100%x2, ETOH 96%, ETOH 80%, ETOH 70% - each for 2Min at RT. 3) IHC protocol... Will Bouin's solution be better ? Thank you all very much in advance Itai M ___ 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 mcaul...@umdnj.edu ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Problem with liver fixation
Thank's all, I'm thinking that my IHC method might be O.K because it is working great with other paraffin embeddedd tissues like diaphragm and various muscles, and (sometimes) also in older liver tissues that i have found in our lab (but i don't know how they were made). I'm using antigen retrieval with 0.01% Citrate buffer, pH 6.0 heated in microwave. but I don't rule out that my IHC method is not optimal for liver tissues, and that there might be a better one. Does someone have a proven fixation, or IHC methods that worked with live tissues ? Thank you all very much Itai M 2010/9/7 Andrea Marion > Hi Itai, > > Liver antigens tend to degrade rapidly, so immediate fixation is > necessary. It sounds like you are getting the tissue into the solution > quickly enough. Fixation occurs more slowly at 4 degrees than room > temperature though - perhaps your antigen is degrading during this > 'slower' fixation? Are you using a sufficient volume of fixative compared > to tissue mass? > > Another concern is that some antigens require paraformaldehyde fixation > (ie a 4% solution of buffered formaldehyde prepared directly from solid > paraformaldehyde). Solutions of formaldehyde marketed as '37% > formaldehyde' or formalin typically contain ~10% methanol added as a > stabilizer, which can interfere with some antigens. For > immunohistochemistry purposes, you may need to prepare your formaldehyde > solution directly from paraformaldehye. See here for more discussion of > formaldehyde solutions: http://publish.uwo.ca/~jkiernan/formglut.htm > > Finally, you list 'IHC protocol', but why are you sure your problem lies > in the tissue fixation/processing and not the staining protocol? You > probably need to do some form of antigen retrieval - what method are you > using? What is your primary antibody dilution? Has your antibody been > validated for use with IHC/IF? Many antibodies simply do not work with > FFPE tissues. There are many other steps that could be causing trouble... > Here is a good beginner's guide for IHC with FFPE tissues if you need: > http://www.abcam.com/ps/pdf/protocols/ihc_p.pdf > > As a side note, your processing steps may be a little long, but I am not > an expert. We use only 30 minutes for each dehydration and clearing step, > and 2 paraffin steps for one hour each with mouse tissue. Good luck! > > Andrea Marion > > amario3 &at& uic &dot& edu > Graduate Student > University of Illinois at Chicago > > > > Rene J Buesa histonet@lists.utsouthwestern.edu , Itai Moshe Bouin's will not do better than what you describe. Are you using antigen retrieval? René J. Patsy Ruegg Itai Moshe itai.mo...@mail.huji.ac.il , I would not fix at 4c, do it at rt. Your fixative looks ok and processing, actually for animal tissues I process for only 20 min a station not 60 min., animal tissues will process quicker because they have less fat, and you can over process them so they become dryed out and hard. About not getting a signal no matter what ab you use, there could be several causes, are you using the proper antigen retrieval technique for that ab, is your detection system matched to your primary antibody, on and on..? Patsy Ruegg, HT(ASCP)QIHC IHCtech 12635 Montview Blvd. Ste.215 Aurora, CO 80045 720-859-4060 fax 720-859-4110 www.ihctech.net www.ihcrg.org --- Itai Moshe אל histonet@lists.utsouthwestern.edu Dear All, I'm trying to use liver sections in paraffin blocks for IHC, but get no signal at all, doesn't matter what antibody i'm using. I'm using section about 3mm thick (the natural thickness of mouse liver) and about 5-7mm Width and Length. My fixation protocol is like this: 1) immediately after killing the mouse i'm putting the sections in a fixation solution that is made from: 10ml formaldehyde 37%, 5ml PBSx20, 85ml DDW - pH 7, 24Hr at 4C. 2) ETOH 70%, ETOH 80%, ETOH 96%, ETOH 100% x2 - each for 1Hr at RT 3) Xylen x2 - each for 1Hr at RT. 4) Paraffin x3 - each at 60C for 1Hr. Before doing IHC: 1) Xylen x2 - each for 10Min at RT 2) ETOH 100%x2, ETOH 96%, ETOH 80%, ETOH 70% - each for 2Min at RT. 3) IHC protocol... Will Bouin's solution be better ? Thank you all very much in advance Itai M ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
