[Histonet] RE: cryostat decontamination
Hi Issac- Our cryostat decon/defrost procedure goes something like this: Remove glass door to cryochamber by pulling up and gently sliding out of track. Allow microtome and chamber to come to room temp. Wipe down exposed surfaces of cryochamber and microtome with 1% bleach or bleach alternative. Wipe dry with paper towels. Spray exposed surfaces with water and wipe dry. Remove cryostat accessories from cryochamber, spraying each (except the brushes) with 1% bleach, then rinsing under RO water. Wipe accessories dry and place in chemical fume hood. Spray thoroughly with absolute alcohol and allow to air dry completely. Disassemble and remove microtome from cryochamber according to manufacturer's instructions found in the owner's manual. Spray microtome and all disassembled parts with 1% bleach and wipe dry with paper towels. Spray with reverse osmosis water and wipe dry with paper towels. Repeat water spray and wipe dry. Place microtome and parts in chemical fume hood. Spray disassembled parts thoroughly with absolute alcohol and allow to air dry completely. Repeat procedure with cryochamber: wipe down with 1% bleach, wipe dry; spray with reverse osmosis water, wipe dry; spray thoroughly with absolute alcohol and allow to air dry completely. Once both cryochamber and microtome are completely dry, reassemble unit according to manufacturer's instructions. Turn main power switch of cryostat on and allow cryostat to reach optimal temperature before cutting sections. If cryostat indicates it needs further drying, turn unit off, use hair dryer to eliminate any moisture and turn on again. Each tech typically cleans up after their turn cutting, by disposing of any shaving, wiping (assembled) knife assembly specimen head, and the areas that collected shavings with 70%ethanol. The external handle of the door and any external control areas used to operate the microtome (ie any areas that gloved hands may have touched) get wiped with a bleach alternative. This is a procedure that our EHS people feel comfortable with given the nature of samples we handle on a daily basis. (and it also wins us points with the manufacturer's Field tech that does our yearly maintenance :) ) Best of luck! ~ Martha Wester Pathology/Lab Manager MedImmune, LLC. One MedImmune Way Gaithersburg, MD 20878 * Message: 4 Date: Sat, 20 Feb 2010 18:38:41 -0800 (PST) From: Isaac O o.isaa...@yahoo.com Subject: [Histonet] CRYOSTAT DECONTAMINATION To: histonet@lists.utsouthwestern.edu Message-ID: 449670.69198...@web111615.mail.gq1.yahoo.com Content-Type: text/plain; charset=iso-8859-1 Hi, I will appreciate it, if you guys could share your procedure for Cryostat Decontamination with me. Thanking you all for your anticipated cooperation. Isaac. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] DRGs
Hi Carol, I have used histogel for these kinds of samples and also other small, thin tissues like insect antennae and insect GI tracts and midguts. Since I get all my projects already fixed in whatever fixative the investigator chooses, rinsed and placed in 70% ETOH the histogel never touches formalin. I don't use formalin on my processor but start in 70%. I've never had a problem with the histogel. We just put the sample in the histogel flat and stand it up (turn 90°) when embedding in paraffin. I use tissue prep for embedding. If you don't want to use histogel you could try to put the drg's on GN Metricel membrane disc filters. We do this with a lot of the samples I receive, actually I have the investigators or their techs do this. The tissue sticks to the membrane and orientation is a dream. The membrane presents no problem when sectioning. You can get it from VWR. Andi Andrea Grantham, HT (ASCP) Senior Research Specialist University of Arizona Cell Biology and Anatomy Histology Service Laboratory P.O.Box 245044 Tucson, AZ 85724 algra...@email.arizona.edu Tel: 520.626.4415 Fax: 520.626.2097 happy slicing and dicing and may all your stains work perfectly - Paula Sicurello P Please consider the environment before printing this email. On Feb 18, 2010, at 11:23 AM, Barone, Carol wrote: Histonetter's...we received a boat-load of mouse DRGs that had been prepared in histogel and are cutting...well..not so good. We normally do DRGs from FS and get beautiful results. We have used histogel before with other small sample and have never had issues... not sure if it is the Histogel or DRG's (fixed in 10% NBF and then transferred to 70% before placed into the histogel).is the issue..I seem to remember that histogel requires formalin and wonder if the transfer to 70% is causing our problem ...but, obviously there is not much room for error with such tiny- tiny samples and they are already process and in paraffin? I am not quite sure how twe can improve the ones that came in histogel, and were processed to paraffin a paraffin blockany idea's? any experience? any anything? Thx- ASAP! cbar...@nemours.org ___ 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] Cryostat decon- what a pain in the butt
Jeff, Do they give any references for the effectiveness of their proposed method? Tim Morken Supervisor, Histology / IPOX UCSF Medical Center San Francisco, CA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jeffrey Silverman Sent: Sunday, February 21, 2010 12:20 PM To: o.isaa...@yahoo.com Cc: histonet@lists.utsouthwestern.edu Subject: [Histonet] Cryostat decon- what a pain in the butt CAP is moving to more rigorous cryostat decontamnation methods - mandating a weekly shutdown and wet chemical disinfection with a tuberculocidal agent for machines used regularly. Now, if a lab is doing 1-3 frozens a week, is that used regularly? We must lobby the CAP for more sensible and practical guidlines. The old wipe down with 70% ETOH without bringing the machine will become non-compliant and useful only as an interim measure. By the way, the UV lamps do not satisfy the CAP standard, I believe. Our system has gone to a commercially available tuberculocidal, virucidal, and broad spectrum bacteriocidal moistened wipes the name of which I will post tomorrow when I get to the job. Here is a skeletonized basic procedure form what CAP will require: 1: Remove all utensils and brush out and collect section debris disposing of this according to regulated medical waste protocol (red bag). 2. Bring the instrument to room temperature. 3: Wipe all working surfaces with the tuberculocidal wipes, visibly moistening all surfaces. Surface must remain wet for 2 minutes. Use multiple wipes as needed. Instruments can be similarly disinfected. 4: Carefully dry all surfaces with gauze. Dispose of all wipes and gauze as biohazardous. 5: Dry and lubricate the microtome as per manufacturer's instructions. 6. Turn on crystat and bring to working temperature. 7. Document procedure on your maintenance log. 8. Look forward to doing it again next week. Look, this is a necessary procedure, but weekly??? Perhaps some workload- based formula or an alert system- like pathologists alert the lab when a case with granulomas or caseating necrosis is sectioned. Every lab will have to bring a tech in on weekends or at night, to do this, or have two cryostats to compensate for the fully one working day most machines will have to be down to be cleaned in this manner. Thoughts? Jeff Silverman ___ 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] Cryostat decon- what a pain in the butt
Jeff, Can you give the reference for the CAP adjustment on decon for crystats please? We have two working cryostats and average 6 to 10 cases (not specimens) a day. This would effectively shut us down for frozens on some days. We have third one that is currently in for repair. Thanks, Pam Marcum UAMS Anatomic Patholgoy Manager - Original Message - From: Tim Morken timothy.mor...@ucsfmedctr.org To: Jeffrey Silverman pathmas...@yahoo.com, o isaac24 o.isaa...@yahoo.com Cc: histonet@lists.utsouthwestern.edu Sent: Monday, February 22, 2010 10:23:17 AM GMT -06:00 US/Canada Central Subject: RE: [Histonet] Cryostat decon- what a pain in the butt Jeff, Do they give any references for the effectiveness of their proposed method? Tim Morken Supervisor, Histology / IPOX UCSF Medical Center San Francisco, CA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jeffrey Silverman Sent: Sunday, February 21, 2010 12:20 PM To: o.isaa...@yahoo.com Cc: histonet@lists.utsouthwestern.edu Subject: [Histonet] Cryostat decon- what a pain in the butt CAP is moving to more rigorous cryostat decontamnation methods - mandating a weekly shutdown and wet chemical disinfection with a tuberculocidal agent for machines used regularly. Now, if a lab is doing 1-3 frozens a week, is that used regularly? We must lobby the CAP for more sensible and practical guidlines. The old wipe down with 70% ETOH without bringing the machine will become non-compliant and useful only as an interim measure. By the way, the UV lamps do not satisfy the CAP standard, I believe. Our system has gone to a commercially available tuberculocidal, virucidal, and broad spectrum bacteriocidal moistened wipes the name of which I will post tomorrow when I get to the job. Here is a skeletonized basic procedure form what CAP will require: 1: Remove all utensils and brush out and collect section debris disposing of this according to regulated medical waste protocol (red bag). 2. Bring the instrument to room temperature. 3: Wipe all working surfaces with the tuberculocidal wipes, visibly moistening all surfaces. Surface must remain wet for 2 minutes. Use multiple wipes as needed. Instruments can be similarly disinfected. 4: Carefully dry all surfaces with gauze. Dispose of all wipes and gauze as biohazardous. 5: Dry and lubricate the microtome as per manufacturer's instructions. 6. Turn on crystat and bring to working temperature. 7. Document procedure on your maintenance log. 8. Look forward to doing it again next week. Look, this is a necessary procedure, but weekly??? Perhaps some workload- based formula or an alert system- like pathologists alert the lab when a case with granulomas or caseating necrosis is sectioned. Every lab will have to bring a tech in on weekends or at night, to do this, or have two cryostats to compensate for the fully one working day most machines will have to be down to be cleaned in this manner. Thoughts? Jeff Silverman ___ 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] DRGs
I think it is strange that we are all doing similar techniques and wind up with different outcomes using the histogel. I would be curious how many of us are using the equipment sold with the histogel for warming and cooling opposed to any of us who don't. we did not purchase the equipment and I wonder sometimes if warming the histogel using other means causes some type of breakdown / and do any of you repeatedly reheat the same tube once it has been warmed and resolidified?? -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrea Grantham Sent: Monday, February 22, 2010 9:41 AM To: HISTONET Subject: Re: [Histonet] DRGs Importance: High Hi Carol, I have used histogel for these kinds of samples and also other small, thin tissues like insect antennae and insect GI tracts and midguts. Since I get all my projects already fixed in whatever fixative the investigator chooses, rinsed and placed in 70% ETOH the histogel never touches formalin. I don't use formalin on my processor but start in 70%. I've never had a problem with the histogel. We just put the sample in the histogel flat and stand it up (turn 90°) when embedding in paraffin. I use tissue prep for embedding. If you don't want to use histogel you could try to put the drg's on GN Metricel membrane disc filters. We do this with a lot of the samples I receive, actually I have the investigators or their techs do this. The tissue sticks to the membrane and orientation is a dream. The membrane presents no problem when sectioning. You can get it from VWR. Andi Andrea Grantham, HT (ASCP) Senior Research Specialist University of Arizona Cell Biology and Anatomy Histology Service Laboratory P.O.Box 245044 Tucson, AZ 85724 algra...@email.arizona.edu Tel: 520.626.4415 Fax: 520.626.2097 happy slicing and dicing and may all your stains work perfectly - Paula Sicurello P Please consider the environment before printing this email. On Feb 18, 2010, at 11:23 AM, Barone, Carol wrote: Histonetter's...we received a boat-load of mouse DRGs that had been prepared in histogel and are cutting...well..not so good. We normally do DRGs from FS and get beautiful results. We have used histogel before with other small sample and have never had issues... not sure if it is the Histogel or DRG's (fixed in 10% NBF and then transferred to 70% before placed into the histogel).is the issue..I seem to remember that histogel requires formalin and wonder if the transfer to 70% is causing our problem ...but, obviously there is not much room for error with such tiny- tiny samples and they are already process and in paraffin? I am not quite sure how twe can improve the ones that came in histogel, and were processed to paraffin a paraffin blockany idea's? any experience? any anything? Thx- ASAP! cbar...@nemours.org ___ 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] DRGs
I also think that it is strange of the way Histogel processes. I have posted on the Histonet previously about this exact problem. I worked with Jennifer Hofecker when she was at Vanderbilt U.(sent her my Histogel and she sent me hers) and ended up with perfectly processed Histogel blocks at our facility and hers. I processed a couple of blocks last week and they were just terrible. No change in the processing schedule, or the way Histogel was liquefied (placed in hot water that was heated in the microwave). Prior to the last two blocks, I must have processed at least a dozen blocks without any problems. There was an incident where I placed two histogels in the same cassete. One processed beautifuly and the other was all shrunken and dried up. I do not liquefy the entire tube, rather I scoop out the approximate amount that I need and transfer to another tube to heat up. If there is anyone out there in Histoland that has not had any issues with the Histogel, can you please post your procedure on liquefying the Histogel, method of cooling/solidifying and processing schedule? The only thing that I do that is not exact is I do not know the temp of my hot water when i place the Histogel to liquefy. I basically have to wait several minutes for the gel to melt and I use it immediately. Any new information or solution from anyone, would be greatly appreciated. Thank you Dusko Trajkovic From: Amy Porter port...@msu.edu To: Andrea Grantham algra...@email.arizona.edu; HISTONET histonet@lists.utsouthwestern.edu Sent: Mon, February 22, 2010 9:01:22 AM Subject: RE: [Histonet] DRGs I think it is strange that we are all doing similar techniques and wind up with different outcomes using the histogel. I would be curious how many of us are using the equipment sold with the histogel for warming and cooling opposed to any of us who don't. we did not purchase the equipment and I wonder sometimes if warming the histogel using other means causes some type of breakdown / and do any of you repeatedly reheat the same tube once it has been warmed and resolidified?? -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrea Grantham Sent: Monday, February 22, 2010 9:41 AM To: HISTONET Subject: Re: [Histonet] DRGs Importance: High Hi Carol, I have used histogel for these kinds of samples and also other small, thin tissues like insect antennae and insect GI tracts and midguts. Since I get all my projects already fixed in whatever fixative the investigator chooses, rinsed and placed in 70% ETOH the histogel never touches formalin. I don't use formalin on my processor but start in 70%. I've never had a problem with the histogel. We just put the sample in the histogel flat and stand it up (turn 90°) when embedding in paraffin. I use tissue prep for embedding. If you don't want to use histogel you could try to put the drg's on GN Metricel membrane disc filters. We do this with a lot of the samples I receive, actually I have the investigators or their techs do this. The tissue sticks to the membrane and orientation is a dream. The membrane presents no problem when sectioning. You can get it from VWR. Andi Andrea Grantham, HT (ASCP) Senior Research Specialist University of Arizona Cell Biology and Anatomy Histology Service Laboratory P.O.Box 245044 Tucson, AZ 85724 algra...@email.arizona.edu Tel: 520.626.4415 Fax: 520.626.2097 happy slicing and dicing and may all your stains work perfectly - Paula Sicurello P Please consider the environment before printing this email. On Feb 18, 2010, at 11:23 AM, Barone, Carol wrote: Histonetter's...we received a boat-load of mouse DRGs that had been prepared in histogel and are cutting...well..not so good. We normally do DRGs from FS and get beautiful results. We have used histogel before with other small sample and have never had issues... not sure if it is the Histogel or DRG's (fixed in 10% NBF and then transferred to 70% before placed into the histogel).is the issue..I seem to remember that histogel requires formalin and wonder if the transfer to 70% is causing our problem ...but, obviously there is not much room for error with such tiny- tiny samples and they are already process and in paraffin? I am not quite sure how twe can improve the ones that came in histogel, and were processed to paraffin a paraffin blockany idea's? any experience? any anything? Thx- ASAP! cbar...@nemours.org ___ 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] Fw: Nail softner
Hi Rene, Could you please fax me your procedure for softening nails, you said it's the best and we do a lot of nails. Thanks in advance. Joyce Wilkinson Fax # 512.9013938 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: cryostat decontamination
Jeff Silverman notes that CAP is moving to more rigorous cryostat decontamination methods - mandating a weekly shutdown and wet chemical disinfection with a tuberculocidal agent for machines used regularly. Cryostats will have to be brought up to room temperature for this procedure. This weekly decontamination routine would take an extraordinary amount of time. In many labs the pathologist would probably be called on to do it, and would probably refuse. I think that a lot of labs will drop CAP accreditation over this issue, unless JCAHO mandates the same routine. I don't know what I would consider an appropriate routine here. It's been a good many years since I cut a frozen section and found granulomatous inflammation suspicious for tuberculosis. (Though I had a paraffin-section case like that last week - don't have the special stain reports yet.) I clearly remember - when I was a resident in one of America's most tuberculous cities forty years ago - a right colon resection for what turned out on frozen section to be not cancer, but tuberculosis involving the lymph nodes around the cecum. I promptly downed the cryostat (no problem, since we had four of them) and had it up to room temperature and was wiping away when the chief (Bill Shelley, of blest memory) came by and observed Boy, do you still believe in the germ theory of disease? Bob Richmond Samurai Pathologist Knoxville TN ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Nail Softeners
Hello Rene, we also do a lot of nails. I was hoping could fax me a copy of your protocol as well. Thank you .215-662-6539 -Fax Albert Santiago, HT(ASCP) Laboratory Manager Dermatopathology 215-662-6008/6539-office 215-662-6150-fax The information contained in this e-mail message is intended only for the personal and confidential use of the recipient(s) named above. If the reader of this message is not the intended recipient or an agent responsible for delivering it to the intended recipient, you are hereby notified that you have received this document in error and that any review, dissemination, distribution, or copying of this message is strictly prohibited. If you have received this communication in error, please notify us immediately by e-mail, and delete the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] tumor bearing perfusion
Hello, We are working on an oncology project that requires the administration of fluorescent dyes in tumor bearing mice. The dyes are allowed to circulate and then tumors are collected, homogenized and centrifuged for fluorescent reading. Specifically, we are looking at the dyes in the interstitial fluid and we cannot have any contamination from blood. We are getting a lot of background fluorescence and we think it may be coming from blood in the tissue. We want to perfuse the animal before tumor collection but we think that if we perfusing with saline will affect the interstitial fluid as well. Has anyone tried to perfuse with something that has high molecular weight? Any advice, tips on this? ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] tumor bearing perfusion
This sounds a lot like what I did for my master's project. What I believe I did to correct for the autofluorescence from RBCs was to have a group of control tumor-bearing mice perfused with saline only, take readings as you would with the dye samples, and in the analysis subtract out the saline readings as background. In theory you should have a similar amount of blood in your saline samples as in your dye samples, if your groups are large enough to account for mouse-to-mouse (or tumor-to-tumor) variability (which I found tended to be rather large...). Regards, Merced Merced M Leiker Research Technician III Cardiovascular Medicine 348 Biomedical Research Building State University of New York at Buffalo 3435 Main St, Buffalo, NY 14214 USA lei...@buffalo.edu 716-829-6118 (Ph) 716-829-2665 (Fx) No trees were harmed in the sending of this email. However, many electrons were severely inconvenienced. --On Monday, February 22, 2010 10:40 AM -0800 Wisam Barkho hermeneu...@gmail.com wrote: Hello, We are working on an oncology project that requires the administration of fluorescent dyes in tumor bearing mice. The dyes are allowed to circulate and then tumors are collected, homogenized and centrifuged for fluorescent reading. Specifically, we are looking at the dyes in the interstitial fluid and we cannot have any contamination from blood. We are getting a lot of background fluorescence and we think it may be coming from blood in the tissue. We want to perfuse the animal before tumor collection but we think that if we perfusing with saline will affect the interstitial fluid as well. Has anyone tried to perfuse with something that has high molecular weight? Any advice, tips on this? ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Merced M Leiker Research Technician III Cardiovascular Medicine 348 Biomedical Research Building State University of New York at Buffalo 3435 Main St, Buffalo, NY 14214 USA lei...@buffalo.edu 716-829-6118 (Ph) 716-829-2665 (Fx) No trees were harmed in the sending of this email. However, many electrons were severely inconvenienced. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Re: EDTA
For many years doing IHC on mouse bones, I have used 10% EDTA, just pHed up to where it goes into solution, with great results for frozen section immunofluorescence. It takes 5 days. For FFPE, we use Richard Allan Decal solution which is HCl and EDTA. Also works great and only takes 1 hour for murine bones. I don't use the acid decal on frozen sections as I find the autofluorescence is too much and I have better antigen preservation with the EDTA for IF. Andrea Hooper --- On Fri, 2/19/10, gayle callis gayle.cal...@bresnan.net wrote: From: gayle callis gayle.cal...@bresnan.net Subject: RE: [Histonet] Re: EDTA To: 'Rittman, Barry R' barry.r.ritt...@uth.tmc.edu, 'Histonet' histonet@lists.utsouthwestern.edu Date: Friday, February 19, 2010, 11:32 PM We use EDTA tetra-sodium salt, pH 7.6 but the pH is adjusted down with glacial acetic acid using continuous stirring and a pH electrode in the solution (titration with the acid). This is a recipe from a Dr. Webster (Webb) S. S. Jee publication. We like this since this 14% EDTA dissolves immediately in water or PBS compared disodium EDTA, and at a higher concentration = more molecules of EDTA available. We are very careful to NOT use this 14% tetrasodium EDTA at it original pH of around 10 or more. Decalcification is still very slow, but a recent bone project required EDTA decalcification (so we thought!) to protect antigens never stained before in our lab. After trying both the EDTA and 10% formic acid, we found the antigen to be very robust after formic acid decalcification too. Lesson learned, don't put off a pilot study to test IHC after acid decalcification. The formic acid would have been faster and cheaper. Gayle Callis -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rittman, Barry R Sent: Friday, February 19, 2010 3:20 PM To: 'Histonet' Subject: RE: [Histonet] Re: EDTA I agree with Gayle and Rene I would not even use a mixture of the two. Formic acid demineralization will work fine, the theory of using EDTA with it makes no sense. EDTA demineralization usually uses the disodium salt. EDTA by itself is not very soluble but its sodium salts are. It can be present as mono-, di-, tri- or tetra-sodium salts depending on the pH. At pH around 11 there are 4 groups that can be replaced with for example calcium. Usually the disodium salt of EDTA is used as the tri and tetrasodium salts are only present in very alkaline solutions that tend to cause tissue maceration. At acidic pH the EDTA will only work slowly and have one group replaceable as the monosodium EDTA. There are several good formulae for using EDTA and formic acid in separate formulae, depending on your specific needs.. Barry From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of gayle callis [gayle.cal...@bresnan.net] Sent: Friday, February 19, 2010 4:03 PM To: 'Histonet' Subject: [Histonet] Re: EDTA You wrote: I am looking into the various decals on the market and have found one that in addition to formic acid, has EDTA in the mix. I have never worked with EDTA so would appre ciate any help in your comments on the use of EDTA in decalcification methods for bone marrow and routine specimens. ** The purpose of EDTA in an acid decalcifier is probably NOT performing the actual removal of calcium (or very little) from the bone but chelating calcium ionized from bone by the acid that then (the calcium) settles to the bottom of the container. I doubt very much that you will be dealing with EDTA but with formic acid decalcification. People who have developed acid decalcifying solutions, with either formic acid or hydrochloric acid tout that the bone section looks very good, better that if EDTA is not present in the solution. I never tested this claim in our bone studies, but it might be something to try, an acid solution containing EDTA and one without EDTA (keeping the same kind of acid in the study). The acid is going to ionize the calcium from bone at a faster rate than the EDTA can chelate the calcium. But most importantly, EDTA does not work in a low pH environment. The chemistry of how EDTA acts at different pHs is well documented by chemists, and found in book chapters on EDTA. My physical chemist spouse supplied me with such a chapter. The pH of formic acid is what, pH 3 or so, and if so, EDTA only begins to chelate calcium around pH 4, and when fully protonated at pH 8, decalcifies faster at that pH than at pH 4, or below pH7. The working pH for most EDTA solutions is commonly 7 to 7.6, but when the pH becomes more alkaline ( and 8 is going into alkaline range) then alkaline sensitive protein linkages can be damaged.
Re: [Histonet] IF staining on peritoneal macrophages
After cytospinning I always dry my slides thoroughly before moving on to a IHC or IF step. I also use Superfrost Plus slides or silane coated slides to increase adherance. I would do that then try various fixatives. You can make a cell pellet as you suggest. Decide ahead of time if you want to fix before embedding (ie: is your antigen preserved in PFA). If so, fix in suspension then spin and resuspend in a small volume of 2% agarose (or Histogel). Then you can embed in OCT for frozen sectioning or wax for FFPE. - Andrea Hooper --- On Thu, 2/18/10, Mauricio Avigdor bitesizell...@gmail.com wrote: From: Mauricio Avigdor bitesizell...@gmail.com Subject: [Histonet] IF staining on peritoneal macrophages To: histonet@lists.utsouthwestern.edu Date: Thursday, February 18, 2010, 11:00 PM Greetings all, I am trying to do immunofluorescence on peritoneal macrophages. I am having a couple of issues that I was hoping one of you could help me resolve. Firstly, I am having uneven results with the Cytospin. Cells tend to get washed off the slides during rinses. Does anyone have tips on how to make cells stick a little better to Cytoslides? Secondly, I have not yet found a satisfactory method for fixation. In order to prevent loss of cells when dipping the slides into fixatives, I am having to air dry the slides before I can do anything to them. I tried the Shandon Collection Fluid (ethanol, isopropanol, carbowax) with great success, but it killed the fluorescence. I am leaning towards spinning the lavage fluid until I get a pellet and then resuspending in PBS with a little BSA added. I hope this makes cells stick well enough that I can put the slides in formalin or acetone. Any thoughts are appreciated! ___ 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] glycogen degeneration???
Hi folks, Has anyone experienced glycogen disappearing from previously excellent control blocks of liver. A glycogen-laden liver should be consistent throughout should it not? (ie we can't cut through it can we??). Or is oxidation a possible culprit here (not heard of it)? Really interested to get some feedback on this one. Thanks so much, Greg Greg Dobbin, R.T. Chief Technologist, Anatomic Pathology Dept. of Laboratory Medicine, Queen Elizabeth Hospital, P.O. Box 6600 Charlottetown, PEC1A 8T5 Phone: (902) 894-2337 Fax: (902) 894-2385 I find that the harder I work, the more luck I seem to have. - Thomas Jefferson - Statement of Confidentiality This message (including attachments) may contain confidential or privileged information intended for a specific individual or organization. If you have received this communication in error, please notify the sender immediately. If you are not the intended recipient, you are not authorized to use, disclose, distribute, copy, print or rely on this email, and should promptly delete this email from your entire computer system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] cryostat decontamination
thank you all for posting about the cryostat decontamination, I have not revised my procedure and we are due for an inspection . I think this is just over kill, our pathologist says we have to do it if cap says. (they also made me throw away all of the dry drys that they said were outdated? now I think they have changed that, I thought that was crazy also.) the way we do it here is if the path thinks it should be down they will tell us. they try to just do smears if it is a know infectious case. I have another question for those who use ventana's benchmark, or I guess anybody that is doing imunos. are you using a universal negative control? or positive and negative with most every case? thanks for your input. everyone have a good day. anita dudley providence hosp mobile alabama _ Hotmail: Free, trusted and rich email service. http://clk.atdmt.com/GBL/go/201469228/direct/01/___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Bleach in autostainer cost problems?
Hi Histonetters, Have you ever had the problem I am having now: light or totally negative staining after service and cleaning the machine with bleach? To explain it better: I have immuno-autostainer from TermoFisher that was working well before I had service from the company. After a representative from the company serviced the machine and cleaned it with bleach, my staining starts to become very light or totally negative on some slides which were running all together on the same day. Unfortunately, I did not realize it immediately and only after several stainings when I expect to see strong staining instead of light like 1+. First of all, I thought it's one of the reagents or my antibody. Then, I checked on the positive slides and finally I came to decision that this is a bleach that was used to clean machine. I can't get in touch with rep almost a week. If you are familiar with this kind of problem, please give me any suggestion. Naira ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] glycogen degeneration???
Air oxidation is the most likely culprit. Will specially show in sections kept at room temperature for more than 3 weeks (as for epitope signal). René J. --- On Mon, 2/22/10, Greg Dobbin gvdob...@ihis.org wrote: From: Greg Dobbin gvdob...@ihis.org Subject: [Histonet] glycogen degeneration??? To: Histonet@lists.utsouthwestern.edu Date: Monday, February 22, 2010, 3:12 PM Hi folks, Has anyone experienced glycogen disappearing from previously excellent control blocks of liver. A glycogen-laden liver should be consistent throughout should it not? (ie we can't cut through it can we??). Or is oxidation a possible culprit here (not heard of it)? Really interested to get some feedback on this one. Thanks so much, Greg Greg Dobbin, R.T. Chief Technologist, Anatomic Pathology Dept. of Laboratory Medicine, Queen Elizabeth Hospital, P.O. Box 6600 Charlottetown, PE C1A 8T5 Phone: (902) 894-2337 Fax: (902) 894-2385 I find that the harder I work, the more luck I seem to have. - Thomas Jefferson - Statement of Confidentiality This message (including attachments) may contain confidential or privileged information intended for a specific individual or organization. If you have received this communication in error, please notify the sender immediately. If you are not the intended recipient, you are not authorized to use, disclose, distribute, copy, print or rely on this email, and should promptly delete this email from your entire computer 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
Re: [Histonet] Bleach in autostainer cost problems?
Bleach is the harshest enemy of any biological reaction and should be avoided as much as possible or absolutely eliminated after is used, otherwise can ruin the best staining protocol. René J. --- On Mon, 2/22/10, Margaryan, Naira nmargar...@childrensmemorial.org wrote: From: Margaryan, Naira nmargar...@childrensmemorial.org Subject: [Histonet] Bleach in autostainer cost problems? To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Date: Monday, February 22, 2010, 3:47 PM Hi Histonetters, Have you ever had the problem I am having now: light or totally negative staining after service and cleaning the machine with bleach? To explain it better: I have immuno-autostainer from TermoFisher that was working well before I had service from the company. After a representative from the company serviced the machine and cleaned it with bleach, my staining starts to become very light or totally negative on some slides which were running all together on the same day. Unfortunately, I did not realize it immediately and only after several stainings when I expect to see strong staining instead of light like 1+. First of all, I thought it's one of the reagents or my antibody. Then, I checked on the positive slides and finally I came to decision that this is a bleach that was used to clean machine. I can't get in touch with rep almost a week. If you are familiar with this kind of problem, please give me any suggestion. 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
Re: [Histonet] frozen sections of cartilage
You should aim at obtaining 4 objectives: 1- extremely thin sections (as thin as you are able to produce); 2- absolutely wrinkle free; 3- using positivey charged slides, and 4- let the sections air dry completely before starting the IHC René J. --- On Mon, 2/22/10, Kim Merriam kmerriam2...@yahoo.com wrote: From: Kim Merriam kmerriam2...@yahoo.com Subject: [Histonet] frozen sections of cartilage To: Histonet histonet@lists.utsouthwestern.edu Date: Monday, February 22, 2010, 2:37 PM 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] histo supervisor
Hello! Does anyone know who the current histology lab supervisor is at Central DuPage Hospital in IL? (the person in charge of hiring?) Thanks! ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] glycogen degeneration???
In addition to Rene's comments, fixation may be an issue. Deeper parts of the block may not be as well fixed as the more superficial parts so the farther into the block you go ... :-( Geoff Greg Dobbin wrote: Hi folks, Has anyone experienced glycogen disappearing from previously excellent control blocks of liver. A glycogen-laden liver should be consistent throughout should it not? (ie we can't cut through it can we??). Or is oxidation a possible culprit here (not heard of it)? Really interested to get some feedback on this one. Thanks so much, Greg Greg Dobbin, R.T. Chief Technologist, Anatomic Pathology Dept. of Laboratory Medicine, Queen Elizabeth Hospital, P.O. Box 6600 Charlottetown, PEC1A 8T5 Phone: (902) 894-2337 Fax: (902) 894-2385 I find that the harder I work, the more luck I seem to have. - Thomas Jefferson - Statement of Confidentiality This message (including attachments) may contain confidential or privileged information intended for a specific individual or organization. If you have received this communication in error, please notify the sender immediately. If you are not the intended recipient, you are not authorized to use, disclose, distribute, copy, print or rely on this email, and should promptly delete this email from your entire computer system. ___ 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] looking for mouse antibodies
Are there good mouse antibodies that will work on dog and cat for the following: Factor 8 GFAP Mycobacterium sp S100a ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC on N-cadherin, CD146, CD144,ELF97 TRAcP.
Hi Julia, I am sorry it's taken me months to reply to this email and hopefully it's not too late. I am only catching up on Histonet messages recently. I stain for VE-cadherin, CD144 in murine bone routinely. RD Systems goat anti-mouse VE-cadherin works very well in paraffin and frozen murine bone. If doing paraffin, I remove femurs/tibias from mice and fix in 4% PFA overnight at 4 deg C (can also fix 2-4h at RT). I decal in Richard Allan decal solution for 1h, sometimes 1.5h if femurs are especially large and remain hard after 1h. Wash well in dH20 and process into paraffin. I antigen retrieve in DAKO Target Retrieval Solution for 10 min at 95-99 degC in veggie steamer with 10 min cool down. Frozen sections for immunofluorescence are fixed in 4% PFA as above. Then decal in 10% EDTA for 5 days. 30% sucrose protect overnight at 4 deg C and snap freeze in OCT. It works well with both protocols. For both frozens and paraffin, primary is at 2-4 ug/ml followed by very specific biotinylated secondary against goat IgG (Jackson Immunoresearch, minimally cross reactive to other species). Then use strep-HRP (Jackson IR) and DAB+ (DAKO) to develop. Please let me know if you need more details. Best, Andrea Hooper --- On Thu, 11/5/09, julia hough julia.m.ho...@hotmail.co.uk wrote: From: julia hough julia.m.ho...@hotmail.co.uk Subject: [Histonet] IHC on N-cadherin, CD146, CD144,ELF97 TRAcP. To: histonet@lists.utsouthwestern.edu Date: Thursday, November 5, 2009, 3:38 PM Dear histonetters. I am trying to perform several new IHC stains on mouse tibia. However, I am having trouble finding literature on some of them. I would like antibodies that work on mouse tissue/bone that are preferably conjugated to a fluorchrome. If anyone has any potentially protocols or information where I could get primary antibodies unconjugated/conjugated for the following that would be lovely: N-cadherin, CD146, CD144,ELF97 TRAcP. Thanks for your time. Julia _ New Windows 7: Simplify what you do everyday. Find the right PC for you. http://www.microsoft.com/uk/windows/buy/___ 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] EDTA
Although what you say is true, it is worth noting that there are some antigens/antibodies more amenable to acid decal. I have experienced this myself several times recently and been burned by thinking EDTA will always give better IHC results. At least for murine bone marrow vasculature antigens, this appears to be the case for some antibodies. Unfortunately as with most things in histology, there is always exceptions to rules of thumb! --- On Fri, 2/19/10, Rene J Buesa rjbu...@yahoo.com wrote: From: Rene J Buesa rjbu...@yahoo.com Subject: Re: [Histonet] EDTA To: 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu, Dorothy LWebb dorothy.l.w...@healthpartners.com Date: Friday, February 19, 2010, 9:03 PM EDTA (which stands for Ethylene-diamino-tratacetic-acid) is a chelating agent and the method of choice to decalcify BM biopsies. The thing is that it should be used alone, and not combined with any acid, as you state (not even formic acid). By itself will produce an extremely gentle decalcification that will be completely suitable for IHC studies. René J. --- On Fri, 2/19/10, Webb, Dorothy L dorothy.l.w...@healthpartners.com wrote: From: Webb, Dorothy L dorothy.l.w...@healthpartners.com Subject: [Histonet] EDTA To: 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu Date: Friday, February 19, 2010, 1:29 PM I am looking into the various decals on the market and have found one that in addition to formic acid, has EDTA in the mix. I have never worked with EDTA so would appre ciate any help in your comments on the use of EDTA in decalcification methods for bone marrow and routine specimens. Thank you ahead of time for your advice! Dorothy Webb, HT (ASCP) Regions Histology Technical Supervisor 651-254-2962 This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this e-mail in error, please immediately notify the HealthPartners Support Center by telephone at (952) 967-6600. You will be reimbursed for reasonable costs incurred in notifying us. HealthPartners R001.0 ___ 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] Bone tissue
We have a difficulty cutting metatarsal bone . It seems that our sections are so dried up. I was thinking that our dehydration have something to do with this which we have placed it in a wrong processing procedure for our large bone. The tissue is 4 mm thick and 1-2 cm in length and width and was dehydrated in 70% - 4 hrs, 80%-4 hrs,95% -4 hrs and 2 changes of 100% 3 hrs each, paraffin is 4 hrs each 2 changes. The tissue was decalcified in 14% EDTA. When we start cutting them it is so brittle and we could not even create a section. I have surfaced decal it and also place in a softener Mollifex some of it work but some does not work. Please help us remedy this tissue. Thank you. Reuel Cornelia, BS MT, AMT Cellular Pathology Texas Scottish Rite Hospital for Children Welborn Street Dallas, TX 75219 Tel: 214-559-7766 fax: 214-559-7768 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Bone tissue
First of all, Mollifex or any other alkaline substance will do nothing useful to the bone. I tend to think that you processed the bone before it was completely decalcified and that is the cause for an incomplete infiltration and a subsequent difficult sectioning. René J. --- On Mon, 2/22/10, Reuel Cornelia reuel.corne...@tsrh.org wrote: From: Reuel Cornelia reuel.corne...@tsrh.org Subject: [Histonet] Bone tissue To: histonet@lists.utsouthwestern.edu Date: Monday, February 22, 2010, 4:36 PM We have a difficulty cutting metatarsal bone . It seems that our sections are so dried up. I was thinking that our dehydration have something to do with this which we have placed it in a wrong processing procedure for our large bone. The tissue is 4 mm thick and 1-2 cm in length and width and was dehydrated in 70% - 4 hrs, 80%-4 hrs,95% -4 hrs and 2 changes of 100% 3 hrs each, paraffin is 4 hrs each 2 changes. The tissue was decalcified in 14% EDTA. When we start cutting them it is so brittle and we could not even create a section. I have surfaced decal it and also place in a softener Mollifex some of it work but some does not work. Please help us remedy this tissue. Thank you. Reuel Cornelia, BS MT, AMT Cellular Pathology Texas Scottish Rite Hospital for Children Welborn Street Dallas, TX 75219 Tel: 214-559-7766 fax: 214-559-7768 ___ 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] DRGs and Histogel
I have had the exact same results, one piece processing well and another in the same cassette shrinking and hardening, when using 1.5-2% low melting point agarose (NuSieve) instead of Histogel. I was thinking of changing to Histogel, but then this thread started last summer and now I see issues continue. I would love to know the best way to do this also! Esther Peters, Ph.D. George Mason University dusko trajkovic wrote: I also think that it is strange of the way Histogel processes. I have posted on the Histonet previously about this exact problem. I worked with Jennifer Hofec ker when she was at Vanderbilt U.(sent her my Histogel and she sent me hers) an d ended up with perfectly processed Histogel blocks at our facility and hers. I processed a couple of blocks last week and they were just terrible. No change in the processing schedule, or the way Histogel was liquefied (placed in hot wa ter that was heated in the microwave). Prior to the last two blocks, I must hav e processed at least a dozen blocks without any problems. There was an incident where I placed two histogels in the same cassete. One processed beautifuly and the other was all shrunken and dried up. I do not liquefy the entire tube, rather I scoop out the approximate amount tha t I need and transfer to another tube to heat up. If there is anyone out there in Histoland that has not had any issues with the Histogel, can you please post your procedure on liquefying the Histogel, method of cooling/solidifying and p rocessing schedule? The only thing that I do that is not exact is I do not know the temp of my hot water when i place the Histogel to liquefy. I basically hav e to wait several minutes for the gel to melt and I use it immediately. Any new information or solution from anyone, would be greatly appreciated. Thank you Dusko Trajkovic From: Amy Porter [1]port...@msu.edu To: Andrea Grantham [2]algra...@email.arizona.edu; HISTONET [3]histo...@list s.utsouthwestern.edu Sent: Mon, February 22, 2010 9:01:22 AM Subject: RE: [Histonet] DRGs I think it is strange that we are all doing similar techniques and wind up with different outcomes using the histogel. I would be curious how many of us are using the equipment sold with the histogel for warming and cooling opposed to any of us who don't. we did not purchase the equipment and I wonder sometimes if warming the histogel using other means causes some type of breakdown / and do any of you repeatedly reheat the same tube once it has been warmed and resolidified?? -Original Message- From: [4]histonet-boun...@lists.utsouthwestern.edu [[5]mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrea Grantham Sent: Monday, February 22, 2010 9:41 AM To: HISTONET Subject: Re: [Histonet] DRGs Importance: High Hi Carol, I have used histogel for these kinds of samples and also other small, thin tissues like insect antennae and insect GI tracts and midguts. Since I get all my projects already fixed in whatever fixative the investigator chooses, rinsed and placed in 70% ETOH the histogel never touches formalin. I don't use formalin on my processor but start in 70%. I've never had a problem with the histogel. We just put the sample in the histogel flat and stand it up (turn 90°) when embedding in paraffin. I use tissue prep for embedding. If you don't want to use histogel you could try to put the drg's on GN Metricel membrane disc filters. We do this with a lot of the samples I receive, actually I have the investigators or their techs do this. The tissue sticks to the membrane and orientation is a dream. The membrane presents no problem when sectioning. You can get it from VWR. Andi Andrea Grantham, HT (ASCP) Senior Research Specialist University of Arizona Cell Biology and Anatomy Histology Service Laboratory P.O.Box 245044 Tucson, AZ 85724 [6]algra...@email.arizona.edu Tel: 520.626.4415Fax: 520.626.2097 happy slicing and dicing and may all your stains work perfectly - Paula Sicurello P Please consider the environment before printing this email. On Feb 18, 2010, at 11:23 AM, Barone, Carol wrote: Histonetter's...we received a boat-load of mouse DRGs that had been prepared in histogel and are cutting...well..not so good. We normally do DRGs from FS and get beautiful results. We have used histogel before with other small sample and have never had issues... not sure if it is the Histogel or DRG's (fixed in 10% NBF and then transferred to 70% before placed into the histogel).is the issue..I seem to remember that histogel requires formalin and wonder if the transfer to 70% is causing our problem ...but, obviously there is not much room for error with such tiny- tiny samples and they are already process and in paraffin? I am not quite sure how twe can improve the ones that came in histogel, and were processed to paraffin a paraffin blockany idea's? any experience? any anything?
[Histonet] Work Load Units
Looking for a good system that works in recording Work Load Units. I've inquired to CAP; they do not have any material available at this time nor recommendations. I've looked at CPT codes but they only reflect billable services in pathology; descriptions are fairly general and cannot be broken down into tasks of the tech. Looked in the Histonet archives; found information not too current. Thanks for any information. Vaughn ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Softening nails
Rene- Can you share your nail softening technique with all of us? Merci. This e-mail may contain confidential or privileged information. If you are not the intended recipient, please advise the sender by reply e-mail and then delete this e-mail immediately. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] best of the nail procedures
The general idea is the extreme base breaks the links in keratin. I tried all of Rene's variations and selected 20% KOH. Well fixed nails (NBF) Soak in 20% KOH for 30-60 minutes (checking at 10 minute intervals) until soft and pliable. (if you let it go to long it turns into mush) Rinse well. Process Embed Cut and stain...PASF works beautifully. May want to run a parallel on split specimens before switching entirely to acclimate. (identifying the end point without having bunches of your specimens turn to mush). Cheryl ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] PPE's embedding and cutting
Usually only if you are working with prions. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of rick.garnh...@memorialhealthsystem.com Sent: Monday, February 22, 2010 6:12 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] PPE's embedding and cutting Anyone in histology land required to wear all PPE's to embed and cut? Rick Garnhart HT(ASCP) Memorial Health System Histology Supervisor 1400 E. Boulder St. Colorado Springs, CO 80909 Cell: 719-365-8357 Ph: 719-365-6926 Fax: 719-365-6373 rick.garnh...@memorialhealthsystem.com Mission: To provide the highest quality health care Vision: To create an outstanding health system where patients heal and people thrive Values: Compassion - Integrity - Quality - Respect - Teamwork www.memorialhealthsystem.com The information contained in or attached to this electronic message is privileged and confidential, intended only for the use of the individual(s) named above. If the reader of this message is not the intended recipient, or the employee or agent responsible to deliver it to the intended recipient, you are hereby notified that any dissemination, distribution, or copying of this communication is strictly prohibited. If you have received this communication in error, please inform the sender immediately and remove any record of this message.___ 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] PPE's embedding and cutting
We wear gloves to section, not necessarily for safety reasons but to eliminate squamous cells on the slides. Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC PO Box 18592 Boulder, Colorado 80308 office (303) 682-3949 fax (303) 682-9060 www.premierlab.com Ship to Address: 1567 Skyway Drive, Unit E Longmont, Colorado 80504 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of rick.garnh...@memorialhealthsystem.com Sent: Monday, February 22, 2010 4:12 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] PPE's embedding and cutting Anyone in histology land required to wear all PPE's to embed and cut? Rick Garnhart HT(ASCP) Memorial Health System Histology Supervisor 1400 E. Boulder St. Colorado Springs, CO 80909 Cell: 719-365-8357 Ph: 719-365-6926 Fax: 719-365-6373 rick.garnh...@memorialhealthsystem.com Mission: To provide the highest quality health care Vision: To create an outstanding health system where patients heal and people thrive Values: Compassion - Integrity - Quality - Respect - Teamwork www.memorialhealthsystem.com The information contained in or attached to this electronic message is privileged and confidential, intended only for the use of the individual(s) named above. If the reader of this message is not the intended recipient, or the employee or agent responsible to deliver it to the intended recipient, you are hereby notified that any dissemination, distribution, or copying of this communication is strictly prohibited. If you have received this communication in error, please inform the sender immediately and remove any record of this message.___ 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] Conference in Australia
Dear Histonetters, The Histotechnology Group of Queensland is holding their conference in conjunction with AIMS (Australian Institute of Medical Scientists) on the weekend of June 11,12, 13. 2010 in Townsville, in tropical Queensland, Australia. This is our winter, but in the tropics winter means sunny days and average maximum temperatures of about 26C (74F) Please explore this website for more detail and note that there is still a call for abstracts current. http://aims.iamevents.com.au/index.php Regards,Laurie. Mr. Laurie REILLY Histopathology School of Veterinary and Biomedical Sciences James Cook University Townsville Qld. 4811 Australia. Phone 07 4781 4468 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Double labeling with antibodies that need different fixatives
Hi all, I would like to do an immunofluorescent double labeling with two antibodies but 1 antibody works on acetone fixed frozen tissue but not on formalin fixed paraffin embedded tissue (CD31 BD pharmingen 553370) and the other one works on formalin fixed paraffin embedded tissue but not on acetone fixed frozen tissue. Is there any way I could still do a double labeling and how? Also, does anyone have experience with zinc fixative? If my antibody works on formalin fixed tissue is it likely to also work on zinc fixed tissue? Thank you very much in advance, Phebe ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Double labeling with antibodies that need different fixatives
Although I haven't tried it myself, others have gotten CD31 from BD to work on FFPE tissue using the tyramide amplification system on zinc buffered formalin fixed sections. I've generally had good luck with zinc buffered formalin myself for many antigens so it may work for your other one. See http://www.ncbi.nlm.nih.gov/pubmed/19052548 Just to be clear, they used zinc buffered formalin, which isn't the same thing as zinc fixative. Adam On Mon, Feb 22, 2010 at 8:41 PM, Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au wrote: Hi all, I would like to do an immunofluorescent double labeling with two antibodies but 1 antibody works on acetone fixed frozen tissue but not on formalin fixed paraffin embedded tissue (CD31 BD pharmingen 553370) and the other one works on formalin fixed paraffin embedded tissue but not on acetone fixed frozen tissue. Is there any way I could still do a double labeling and how? Also, does anyone have experience with zinc fixative? If my antibody works on formalin fixed tissue is it likely to also work on zinc fixed tissue? Thank you very much in advance, Phebe ___ 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: Re: [Histonet] Double labeling with antibodies that need differentfixatives
what is the other marker than CD31? May you let us know? 2010-02-23 TF 发件人： Adam . 发送时间： 2010-02-23 10:55:18 收件人： Phebe Verbrugghe 抄送： histonet 主题： Re: [Histonet] Double labeling with antibodies that need differentfixatives Although I haven't tried it myself, others have gotten CD31 from BD to work on FFPE tissue using the tyramide amplification system on zinc buffered formalin fixed sections. I've generally had good luck with zinc buffered formalin myself for many antigens so it may work for your other one. See http://www.ncbi.nlm.nih.gov/pubmed/19052548 Just to be clear, they used zinc buffered formalin, which isn't the same thing as zinc fixative. Adam On Mon, Feb 22, 2010 at 8:41 PM, Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au wrote: Hi all, I would like to do an immunofluorescent double labeling with two antibodies but 1 antibody works on acetone fixed frozen tissue but not on formalin fixed paraffin embedded tissue (CD31 BD pharmingen 553370) and the other one works on formalin fixed paraffin embedded tissue but not on acetone fixed frozen tissue. Is there any way I could still do a double labeling and how? Also, does anyone have experience with zinc fixative? If my antibody works on formalin fixed tissue is it likely to also work on zinc fixed tissue? Thank you very much in advance, Phebe ___ 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] Double labeling with antibodies that need different fixatives
Hello Adam, Thank you very much for this very useful information! Do you know whether this would also work on tissue fixed with formalin instead of zinc buffered formalin by any chance? Also, could you give me the recipe for the zinc formalin and can I use a standard tissue processor for embedding in paraffin or should I use a specific protocol manually and if so, which? Thanks! Phebe From: Adam . [mailto:anonwu...@gmail.com] Sent: Tuesday, 23 February 2010 10:52 AM To: Phebe Verbrugghe Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Double labeling with antibodies that need different fixatives Although I haven't tried it myself, others have gotten CD31 from BD to work on FFPE tissue using the tyramide amplification system on zinc buffered formalin fixed sections. I've generally had good luck with zinc buffered formalin myself for many antigens so it may work for your other one. See http://www.ncbi.nlm.nih.gov/pubmed/19052548 Just to be clear, they used zinc buffered formalin, which isn't the same thing as zinc fixative. Adam On Mon, Feb 22, 2010 at 8:41 PM, Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au wrote: Hi all, I would like to do an immunofluorescent double labeling with two antibodies but 1 antibody works on acetone fixed frozen tissue but not on formalin fixed paraffin embedded tissue (CD31 BD pharmingen 553370) and the other one works on formalin fixed paraffin embedded tissue but not on acetone fixed frozen tissue. Is there any way I could still do a double labeling and how? Also, does anyone have experience with zinc fixative? If my antibody works on formalin fixed tissue is it likely to also work on zinc fixed tissue? Thank you very much in advance, Phebe ___ 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] Double labeling with antibodies that need different fixatives
I have no idea if it would work with regular formalin. In my experience, many antigens work as well in zinc buffered formalin without any antigen retrieval as regular formalin with antigen retrieval. But really, you just have to try it yourself. On antibodies I've gotten this to work, I use commercially available zinc buffered formalin which comes in gallon jugs for around $50. We don't do any special processing. We plop our sample (bones) in zinc formalin overnight at 4C, decalcify in EDTA or formic acid (only necessary for bones), and then embed just like any other sample. Adam On Mon, Feb 22, 2010 at 9:45 PM, Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au wrote: Hello Adam, Thank you very much for this very useful information! Do you know whether this would also work on tissue fixed with formalin instead of zinc buffered formalin by any chance? Also, could you give me the recipe for the zinc formalin and can I use a standard tissue processor for embedding in paraffin or should I use a specific protocol manually and if so, which? Thanks! Phebe -- *From:* Adam . [mailto:anonwu...@gmail.com] *Sent:* Tuesday, 23 February 2010 10:52 AM *To:* Phebe Verbrugghe *Cc:* histonet@lists.utsouthwestern.edu *Subject:* Re: [Histonet] Double labeling with antibodies that need different fixatives Although I haven't tried it myself, others have gotten CD31 from BD to work on FFPE tissue using the tyramide amplification system on zinc buffered formalin fixed sections. I've generally had good luck with zinc buffered formalin myself for many antigens so it may work for your other one. See http://www.ncbi.nlm.nih.gov/pubmed/19052548 Just to be clear, they used zinc buffered formalin, which isn't the same thing as zinc fixative. Adam On Mon, Feb 22, 2010 at 8:41 PM, Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au wrote: Hi all, I would like to do an immunofluorescent double labeling with two antibodies but 1 antibody works on acetone fixed frozen tissue but not on formalin fixed paraffin embedded tissue (CD31 BD pharmingen 553370) and the other one works on formalin fixed paraffin embedded tissue but not on acetone fixed frozen tissue. Is there any way I could still do a double labeling and how? Also, does anyone have experience with zinc fixative? If my antibody works on formalin fixed tissue is it likely to also work on zinc fixed tissue? Thank you very much in advance, Phebe ___ 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] Double labeling with antibodies that need different fixatives
Hi Adam, Thanks a lot, might just give it a go on just formalin fixed tissue first. Phebe From: Adam . [mailto:anonwu...@gmail.com] Sent: Tuesday, 23 February 2010 12:01 PM To: Phebe Verbrugghe Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Double labeling with antibodies that need different fixatives I have no idea if it would work with regular formalin. In my experience, many antigens work as well in zinc buffered formalin without any antigen retrieval as regular formalin with antigen retrieval. But really, you just have to try it yourself. On antibodies I've gotten this to work, I use commercially available zinc buffered formalin which comes in gallon jugs for around $50. We don't do any special processing. We plop our sample (bones) in zinc formalin overnight at 4C, decalcify in EDTA or formic acid (only necessary for bones), and then embed just like any other sample. Adam On Mon, Feb 22, 2010 at 9:45 PM, Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au wrote: Hello Adam, Thank you very much for this very useful information! Do you know whether this would also work on tissue fixed with formalin instead of zinc buffered formalin by any chance? Also, could you give me the recipe for the zinc formalin and can I use a standard tissue processor for embedding in paraffin or should I use a specific protocol manually and if so, which? Thanks! Phebe From: Adam . [mailto:anonwu...@gmail.com] Sent: Tuesday, 23 February 2010 10:52 AM To: Phebe Verbrugghe Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Double labeling with antibodies that need different fixatives Although I haven't tried it myself, others have gotten CD31 from BD to work on FFPE tissue using the tyramide amplification system on zinc buffered formalin fixed sections. I've generally had good luck with zinc buffered formalin myself for many antigens so it may work for your other one. See http://www.ncbi.nlm.nih.gov/pubmed/19052548 Just to be clear, they used zinc buffered formalin, which isn't the same thing as zinc fixative. Adam On Mon, Feb 22, 2010 at 8:41 PM, Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au wrote: Hi all, I would like to do an immunofluorescent double labeling with two antibodies but 1 antibody works on acetone fixed frozen tissue but not on formalin fixed paraffin embedded tissue (CD31 BD pharmingen 553370) and the other one works on formalin fixed paraffin embedded tissue but not on acetone fixed frozen tissue. Is there any way I could still do a double labeling and how? Also, does anyone have experience with zinc fixative? If my antibody works on formalin fixed tissue is it likely to also work on zinc fixed tissue? Thank you very much in advance, Phebe ___ 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: RE: [Histonet] Double labeling with antibodies that need differentfixatives
From:Anatech Ltd. anat...@net-link.net To:histo...@pathology.swmed.edu Reply-To: Date:Mon, 21 Jun 1999 12:12:19 -0400 Content-Type:text/plain; charset=us-ascii A comprehensive review of zinc formalin up to 1992 appears in the following paper: RW Dapson, 1993. Fixation for the 1990's: a review of needs and accomplishments. Biotechnic Histochem 68:75-82. The mechanism appears to be that zinc ions hold macromolecules in their native conformation via coordinate bonds, preventing the damaging crosslinkages that formaldehyde alone would create. The result is greatly enhanced immunopreservation: rarely is antigen retrieval necessary, and primary antibodies can be diluted 2-10 fold greater than usual. The original use of zinc formalin (by P. Banks and coworkers) was to prevent nuclear bubbling artifact in tissues fixed for less than 24-48 hours. Because nuclear morphology is so sharply defined, zinc formalin is frequently used as a replacement for B-5. Today there are several distinct varieties of zinc formalin, but all share the advantages mentioned above. The original formlula for zinc formalin is 1% zinc sulfate heptahydrate in 10% unbuffered formalin. It tends to precipitate in processors, as the zinc is not soluble in the 70% alcohol used in the first dehydration station. This can cause lines to plug if routine acidic rinses are not performed. Neutral or alkaline water will not dissolve the precipitate. Some processor manufacturers do not want zinc formalin on their machines because of this. The zinc inside the specimens also precipitates (you cannot see it), and causes difficulties in microtomy. Anatech Ltd. developed an unbuffered zinc sulfate formula that does not precipitate in 70% alcohol (it will precipitate if you go directly from it to 80% or higher alcohol), and can be used in all processors. Tissues are not crunchy, and in fact customers often remark that they are easier to cut than those fixed in NBF. All unbuffered zinc formalins are acidic (pH varies from about 3 up to about 4.5). Formalin pigment will develop below about pH 5.3, faster as pH gets lower. The vast majority of our customers using unbuffered zinc formalin do not report formalin pigment artifact (which frankly surprised us), and I can only assume that it is because exposure time is too short for it to happen. Given comparable pH levels, acidic zinc formalin and acidic (unbuffered) formalin will behave similarly regarding pigment formation. Buffered zinc formalin is also available, but again, some formulations precipitate badly in processors. Anatech's product (Z-Fix) is freely soluble in alcohol, and can be made as an alcoholic buffered zinc formalin for those of you who like the added benefits (speed, penetration of fat) that alcoholic formalin provides. Most zinc formalin solutions do not corrode metal any faster than formaldehyde will. Remember that formaldehyde is rather corrosive to nearly all steel, including most forms of stainless (316 and 410 stainless are safe). I suspect that Pearl Gervais' cassette lids are made of some other stainless steel; 316 and 410 are expensive and notoriously difficult to mill. One group of zinc formalin solutions is highly corrosive, however, and that is made with zinc chloride. Faster than its cousins and nearly as aggressive as mercuric chloride, it has been used (not by us) as a B-5 replacement. It can overfix tissues, just like B-5 does, and absolutely must not be put in an enclosed processor. There are very effective rapid, zinc-based B-5 replacements that do not contain the chloride salt. Anatech's products are availble only in the US and Canada. Shandon Lipshaw has a processor-compatible zinc formalin (unbufferd) that is available worldwide. I believe that Richard-Allan's zinc formalin product line is available only in the US (Joan, correct me if I err here!). Sorry for the lengthy answer: I wanted to cover everyone's questions and concerns. Dick Richard W. Dapson, Ph.D. ANATECH LTD. 1020 Harts Lake Road Battle Creek, MI 49015 800-262-8324 or 616-964-6450 Fax 616-964-8084 E-mail anat...@net-link.net http://www.net-link.net/anatech 2010-02-23 TF 发件人： Phebe Verbrugghe 发送时间： 2010-02-23 11:50:40 收件人： Adam . 抄送： histonet 主题： RE: [Histonet] Double labeling with antibodies that need differentfixatives Hello Adam, Thank you very much for this very useful information! Do you know whether this would also work on tissue fixed with formalin instead of zinc buffered formalin by any chance? Also, could you give me the recipe for the zinc formalin and can I use a standard tissue processor for embedding in paraffin or should I use a specific protocol manually and if so, which? Thanks! Phebe From: Adam . [mailto:anonwu...@gmail.com] Sent: Tuesday, 23 February 2010 10:52 AM To: Phebe Verbrugghe Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Double labeling with antibodies that need different
RE: [Histonet] Double labeling with antibodies that need differentfixatives
Hi Phebe The first place to look when working up Antibodies is the Specification sheet from the manufacturer. If you look at the BD sheet for their CD31 Immunohistochemistry is recommended for Zinc fixed paraffin sections and acetone fixed frozeb sections but not recommended for formalin fixed paraffin sections. regards Tony Tony Reilly Chief Scientist, Anatomical Pathology Pathology Queensland-PA Laboratory _ Clinical and Statewide Services Division| QueenslandHealth Level 1, Building 15,Princess Alexandra Hospital Ipswich Road,WOOLLOONGABBA Qld4102 Ph: 07 3176 2412 Mob: 0402 139411 Fax: 07 3176 2930 Email: tony_rei...@health.qld.gov.au Web: www.health.qld.gov.au/qhcss/ Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au 23/02/2010 2:09 pm Hi Adam, Thanks a lot, might just give it a go on just formalin fixed tissue first. Phebe From: Adam . [mailto:anonwu...@gmail.com] Sent: Tuesday, 23 February 2010 12:01 PM To: Phebe Verbrugghe Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Double labeling with antibodies that need different fixatives I have no idea if it would work with regular formalin. In my experience, many antigens work as well in zinc buffered formalin without any antigen retrieval as regular formalin with antigen retrieval. But really, you just have to try it yourself. On antibodies I've gotten this to work, I use commercially available zinc buffered formalin which comes in gallon jugs for around $50. We don't do any special processing. We plop our sample (bones) in zinc formalin overnight at 4C, decalcify in EDTA or formic acid (only necessary for bones), and then embed just like any other sample. Adam On Mon, Feb 22, 2010 at 9:45 PM, Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au wrote: Hello Adam, Thank you very much for this very useful information! Do you know whether this would also work on tissue fixed with formalin instead of zinc buffered formalin by any chance? Also, could you give me the recipe for the zinc formalin and can I use a standard tissue processor for embedding in paraffin or should I use a specific protocol manually and if so, which? Thanks! Phebe From: Adam . [mailto:anonwu...@gmail.com] Sent: Tuesday, 23 February 2010 10:52 AM To: Phebe Verbrugghe Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Double labeling with antibodies that need different fixatives Although I haven't tried it myself, others have gotten CD31 from BD to work on FFPE tissue using the tyramide amplification system on zinc buffered formalin fixed sections. I've generally had good luck with zinc buffered formalin myself for many antigens so it may work for your other one. See http://www.ncbi.nlm.nih.gov/pubmed/19052548 Just to be clear, they used zinc buffered formalin, which isn't the same thing as zinc fixative. Adam On Mon, Feb 22, 2010 at 8:41 PM, Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au wrote: Hi all, I would like to do an immunofluorescent double labeling with two antibodies but 1 antibody works on acetone fixed frozen tissue but not on formalin fixed paraffin embedded tissue (CD31 BD pharmingen 553370) and the other one works on formalin fixed paraffin embedded tissue but not on acetone fixed frozen tissue. Is there any way I could still do a double labeling and how? Also, does anyone have experience with zinc fixative? If my antibody works on formalin fixed tissue is it likely to also work on zinc fixed tissue? Thank you very much in advance, Phebe ___ 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, including any attachments sent with it, is confidential and for the sole use of the intended recipient(s). This confidentiality is not waived or lost, if you receive it and you are not the intended recipient(s), or if it is transmitted/received in error. Any unauthorised use, alteration, disclosure, distribution or review of this email is strictly prohibited. The information contained in this email, including any attachment sent with it, may be subject to a statutory duty of confidentiality if it relates to health service matters. If you are not the intended recipient(s), or if you have received this email in error, you are asked to immediately notify the sender by telephone collect on Australia +61 1800 198 175 or by return email. You should also delete this email, and any copies, from your computer system network and destroy any hard copies produced. If not an intended recipient of this email, you must not copy,
RE: [Histonet] Double labeling with antibodies that need differentfixatives
Hi Phebe The first place to look when working up Antibodies is the Specification sheet from the manufacturer. If you look at the BD sheet for their CD31 Immunohistochemistry is recommended for Zinc fixed paraffin sections and acetone fixed frozeb sections but not recommended for formalin fixed paraffin sections. regards Tony Tony Reilly Chief Scientist, Anatomical Pathology Pathology Queensland-PA Laboratory _ Clinical and Statewide Services Division| QueenslandHealth Level 1, Building 15,Princess Alexandra Hospital Ipswich Road,WOOLLOONGABBA Qld4102 Ph: 07 3176 2412 Mob: 0402 139411 Fax: 07 3176 2930 Email: tony_rei...@health.qld.gov.au Web: www.health.qld.gov.au/qhcss/ Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au 23/02/2010 2:09 pm Hi Adam, Thanks a lot, might just give it a go on just formalin fixed tissue first. Phebe From: Adam . [mailto:anonwu...@gmail.com] Sent: Tuesday, 23 February 2010 12:01 PM To: Phebe Verbrugghe Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Double labeling with antibodies that need different fixatives I have no idea if it would work with regular formalin. In my experience, many antigens work as well in zinc buffered formalin without any antigen retrieval as regular formalin with antigen retrieval. But really, you just have to try it yourself. On antibodies I've gotten this to work, I use commercially available zinc buffered formalin which comes in gallon jugs for around $50. We don't do any special processing. We plop our sample (bones) in zinc formalin overnight at 4C, decalcify in EDTA or formic acid (only necessary for bones), and then embed just like any other sample. Adam On Mon, Feb 22, 2010 at 9:45 PM, Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au wrote: Hello Adam, Thank you very much for this very useful information! Do you know whether this would also work on tissue fixed with formalin instead of zinc buffered formalin by any chance? Also, could you give me the recipe for the zinc formalin and can I use a standard tissue processor for embedding in paraffin or should I use a specific protocol manually and if so, which? Thanks! Phebe From: Adam . [mailto:anonwu...@gmail.com] Sent: Tuesday, 23 February 2010 10:52 AM To: Phebe Verbrugghe Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Double labeling with antibodies that need different fixatives Although I haven't tried it myself, others have gotten CD31 from BD to work on FFPE tissue using the tyramide amplification system on zinc buffered formalin fixed sections. I've generally had good luck with zinc buffered formalin myself for many antigens so it may work for your other one. See http://www.ncbi.nlm.nih.gov/pubmed/19052548 Just to be clear, they used zinc buffered formalin, which isn't the same thing as zinc fixative. Adam On Mon, Feb 22, 2010 at 8:41 PM, Phebe Verbrugghe pverbrug...@meddent.uwa.edu.au wrote: Hi all, I would like to do an immunofluorescent double labeling with two antibodies but 1 antibody works on acetone fixed frozen tissue but not on formalin fixed paraffin embedded tissue (CD31 BD pharmingen 553370) and the other one works on formalin fixed paraffin embedded tissue but not on acetone fixed frozen tissue. Is there any way I could still do a double labeling and how? Also, does anyone have experience with zinc fixative? If my antibody works on formalin fixed tissue is it likely to also work on zinc fixed tissue? Thank you very much in advance, Phebe ___ 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, including any attachments sent with it, is confidential and for the sole use of the intended recipient(s). This confidentiality is not waived or lost, if you receive it and you are not the intended recipient(s), or if it is transmitted/received in error. Any unauthorised use, alteration, disclosure, distribution or review of this email is strictly prohibited. The information contained in this email, including any attachment sent with it, may be subject to a statutory duty of confidentiality if it relates to health service matters. If you are not the intended recipient(s), or if you have received this email in error, you are asked to immediately notify the sender by telephone collect on Australia +61 1800 198 175 or by return email. You should also delete this email, and any copies, from your computer system network and destroy any hard copies produced. If not an intended recipient of this email, you must not copy,
