[Histonet] accidentally frozen samples
One of our students accidentally put his samples in the freezer after formaldehyde/glutardialdehyde/PBS fixation. The samples (fish very early stage, of course rare species) were meant for LM/EM morphology studies. Good morphology is out, but is there a chance he can do cartilage/bone staining? Or do we need to send him abroad for new sampling at the next spawning time? Tora Bardal Senior Engineer NTNU Center of Fisheries and Aquaculture ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] accidentally frozen samples
Probably the bone staining will be OK - how about sending the student to the arctic circle for a few days in winter? - that may be a good lesson about not freezing tissues. Just kidding :-) On Fri, May 20, 2011 at 9:24 AM, Tora Bardal tora.bar...@bio.ntnu.nowrote: One of our students accidentally put his samples in the freezer after formaldehyde/glutardialdehyde/PBS fixation. The samples (fish very early stage, of course rare species) were meant for LM/EM morphology studies. Good morphology is out, but is there a chance he can do cartilage/bone staining? Or do we need to send him abroad for new sampling at the next spawning time? Tora Bardal Senior Engineer NTNU Center of Fisheries and Aquaculture ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Louise Renton Bone Research Unit University of the Witwatersrand Johannesburg South Africa +27 11 717 2298 (tel fax) 073 5574456 (emergencies only) There are nights when the wolves are silent and only the moon howls. George Carlin No trees were killed in the sending of this message. However, many electrons were terribly inconvenienced. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Histology survey
Histology Colleagues, I am interested in collecting information to better understand what motivates Histotechnologists in the workplace as well as job satisfaction levels within our field. This ten question survey should just take a few minutes to complete and survey responses will be kept in strict confidence. You are under no obligation to finish the survey and may stop at any time. I am currently looking at data specific to the United States within the clinical setting. Please do not complete this survey if you live outside the United States or if you currently work in a research setting. Here is the survey link: http://www.surveymonkey.com/s/YTX97M8 Please note: this survey will close on May 23rd or when 100 responses are received, whichever occurs first. If clicking on the link does not work, please copy and paste the link into your browser. I would appreciate if you would also forward this opportunity to other histologists you know who may be interested in participating. I would be willing to share the results with whoever is interested. Thank you for your help with this survey. Leslie Chaussey, B.S., HT (ASCP) lcha...@yahoo.com This message and any included attachments are intended only for the addressee. The information contained in this message is confidential and may constitute proprietary or non-public information under international, federal, or state laws. Unauthorized forwarding, printing, copying, distribution, or use of such information is strictly prohibited and may be unlawful. If you are not the addressee, please promptly delete this message and notify the sender of the delivery error by e-mail. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC pos. neg. control question
Pete, OK, time for an example: A pathologist orders 4 IHC's on a block. I run 5 slides total: 4 IHC slides with a section of patient tissue and a known positive. 1 slide with patient tissue only for the negative control. The one negative control is put through the retrieval/protocol that is most likely to cause nonspecific staining. I don't run the known positive control tissue used on the 4 actual IHC slides as negative controls. Perhaps I didn't point out that my original post was addressing the negative control? I'm a bit surprised by the confusion. As for the question of how I know the staining seen in a positive control is truly positive?: All known positive controls are tested previously so I know that they work for the antibody that I'm using them for and I would assume the pathologist uses morphology and localization of staining to determine that the positive control is working. That's what I do. I've gone through 7 CAP inspections utilizing the practices above with no problems thus far. Perhaps you could enlighten me on IHC requirements that I haven't come across. Glen D. -Original Message- From: pete.peder...@healthonecares.com [mailto:pete.peder...@healthonecares.com] Sent: Thursday, May 19, 2011 2:31 PM To: Dawson, Glen; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately. Curt ___ 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
RE: [Histonet] IHC pos. neg. control question
Tj, Amen brother! GD -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Thomas Jasper Sent: Thursday, May 19, 2011 2:39 PM To: pete.peder...@healthonecares.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question Pete, When you run a positive control. The tissue is already a known positive (or it should be) for whichever antibody you are running regardless of prior handling. It would be impossible for this not to be so. However, with a negative, the concern is seeing how the patient tissue turns out when subjected to all the same conditions, minus the antibody. tj -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of pete.peder...@healthonecares.com Sent: Thursday, May 19, 2011 12:31 PM To: gdaw...@dynacaremilwaukee.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately. Curt ___ 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 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] Denatured Alcohol
Q#1 - yes, it is still 200 proof Q#2 - no it should be not considered as a controlled substance. If you still have to keep a log of its use = bureaucratic ignorance and lack of adaptability, like the case in the British army were the artillery kept in the personnel assigned to each movable gun 2 soldiers to restrain the horses that pulled the piece, even years after there were no more horses pulling the piece. René J. From: Joe Nocito jnoc...@satx.rr.com To: Histonet histonet@lists.utsouthwestern.edu Sent: Thursday, May 19, 2011 7:13 PM Subject: [Histonet] Denatured Alcohol Greetings and Salutations Histoland, I have a question about denatured alcohol. I work in a government facility and absolute alcohol (200 proof) is still considered a controlled substance. This requires a monthly inventory by someone from another department. Years ago (ok many, many years ago) I remember that 200 proof had an IRS sticker covering the cap. The alcohol we have is denatured alcohol, 200 proof, which according to the MSDS is cut with kerosene. There is no IRS sticker on the bottles. Question #1- If the alcohol is cut with something other than ethanol, ( which usually it's cut with methanol) is it still 200 proof? Question #2- If it is denatured, it is considered not suitable for drinking. If the substance is not suitable for drinking, then why would it be considered a controlled substance? See my dilemma? We would like to get it removed from our controlled substance list, but we need a reliable source. The company (whom shall remain nameless because of my past history of inflaming vendors) was useless. I don't think the government accepts Wikipedia as an authoritative source. Thanks JTT ___ 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] accidentally frozen samples
The bast approach will be to try to do the cartilage/bone staining. It is preferable to do this than to consider the samples already ruined and incur in the expense of a new sampling. René J. From: Tora Bardal tora.bar...@bio.ntnu.no To: histonet@lists.utsouthwestern.edu Sent: Friday, May 20, 2011 3:24 AM Subject: [Histonet] accidentally frozen samples One of our students accidentally put his samples in the freezer after formaldehyde/glutardialdehyde/PBS fixation. The samples (fish very early stage, of course rare species) were meant for LM/EM morphology studies. Good morphology is out, but is there a chance he can do cartilage/bone staining? Or do we need to send him abroad for new sampling at the next spawning time? Tora Bardal Senior Engineer NTNU Center of Fisheries and Aquaculture ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC pos. neg. control question
And the block in question has already been proven positive using THAT procedure and antibody during validation. Claire From: histonet-boun...@lists.utsouthwestern.edu on behalf of Thomas Jasper Sent: Thu 5/19/2011 2:39 PM To: pete.peder...@healthonecares.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question Pete, When you run a positive control. The tissue is already a known positive (or it should be) for whichever antibody you are running regardless of prior handling. It would be impossible for this not to be so. However, with a negative, the concern is seeing how the patient tissue turns out when subjected to all the same conditions, minus the antibody. tj -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of pete.peder...@healthonecares.com Sent: Thursday, May 19, 2011 12:31 PM To: gdaw...@dynacaremilwaukee.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately. Curt ___ 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 mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu
RE: [Histonet] IHC pos. neg. control question
All very insightful input. I took the liberty of forwarding some of the comments to the client, I think I errantly sent some of the critical comments about him too. I'll probably hear about it later, the best was the clown residency, I was in tears laughing. i'll let you all know what he says about CAPs protocol, don't exactly know how he can argue against that. Thanks all, Curt -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Ingles Claire Sent: Friday, May 20, 2011 6:30 AM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question And the block in question has already been proven positive using THAT procedure and antibody during validation. Claire From: histonet-boun...@lists.utsouthwestern.edu on behalf of Thomas Jasper Sent: Thu 5/19/2011 2:39 PM To: pete.peder...@healthonecares.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question Pete, When you run a positive control. The tissue is already a known positive (or it should be) for whichever antibody you are running regardless of prior handling. It would be impossible for this not to be so. However, with a negative, the concern is seeing how the patient tissue turns out when subjected to all the same conditions, minus the antibody. tj -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of pete.peder...@healthonecares.com Sent: Thursday, May 19, 2011 12:31 PM To: gdaw...@dynacaremilwaukee.com; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question Glen, If I am to understand you correctly you are saying control tissue is not treated the same as patient tissue, therefore is useless as a negative control correct? Then inversely doesn't that mean the same thing towards the use of a positive control? How can you guarantee the positive control tissue was treated the same as the positive stained patient tissue? According to your logic it cannot. Therefore, without the use of a negative control how can you say the staining seen in the positive control is truly positive and not artifact? Best practice says use positive and negative patient and control tissue. Please enlighten me if you know anything to the contrary? Pete Pedersen B.S. HTL (ASCP) Anatomic Pathology Supervisor -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawson, Glen Sent: Thursday, May 19, 2011 12:32 PM To: histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] IHC pos. neg. control question IMHO: Running any piece of tissue as a control that does not belong to the patient being tested makes zero sense. Because it would not be from the patient tissue being tested, how do you know if it was handled the same as the patient tissue? For example: 1) Were they processed the same way? 2) Did the patient tissue dry out in the OR before it was delievered? 3) Was the patient tissue ever irradiated? 4) Does the patient tissue contain any of a number of substances that could cause non-specific staining. 5) Was the patient abducted by aliens? My point is that running a piece of tissue as a negative control that is not even from the patient being tested throws all of the conditions that the patient tissue was exposed to prior to and during processing out the window. This makes NO sense. Glen Dawson BS, HT(ASCP) QIHC IHC Manager Milwaukee, WI -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt Tague Sent: Thursday, May 19, 2011 11:04 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. neg. control question I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg. control. Thanks for your guidance. Email: I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control. The patient's own tissue cannot be used as a negative control. The tissue that stained positively must serve as the negative control without the antibody. This is critical and you need to correct that immediately. Curt ___ Histonet mailing list Histonet@lists.utsouthwestern.edu
[Histonet] Re: Accidental freezing of fish samples
You wrote: Probably the bone staining will be OK - how about sending the student to the arctic circle for a few days in winter? - that may be a good lesson about not freezing tissues. Just kidding :-) On Fri, May 20, 2011 at 9:24 AM, Tora Bardal tora.bardal http://lists.utsouthwestern.edu/mailman/listinfo/histonet @t bio.ntnu.nowrote: One of our students accidentally put his samples in the freezer after formaldehyde/glutaraldehyde/PBS fixation. The samples (fish very early stage, of course rare species) were meant for LM/EM morphology studies. Good morphology is out, but is there a chance he can do cartilage/bone staining? Or do we need to send him abroad for new sampling at the next spawning time? Tora Bardal Senior Engineer NTNU Center of Fisheries and Aquaculture I was amused by Louise's reply. Your comment good morphology is out is correct, especially for EM where bone and cartilage may NOT be optimal after a freeze/ thaw. EM requires optimal fixation and handling to have the best results. There will probably be too much freezing artifact damage (large water ice crystal formation) in the EM samples. However, if doing light microscopy and not worried about soft tissues and with paraffin processing , the bone and cartilage may be ok, but not entirely be ideal ..individual bone and cartilage cells, along with soft tissues, may still show the effects of freezing artifact. These effects may be seen more in cartilage with its higher water content, but give the LM samples a try. You may salvage part of the study. Then send the student out for samples and insist he/she reads up on and understand the damage freezing/thawing can have on fixed samples. I suggest, in lieu of sending your student to the Arctic, that he/she reads the excellent discussion of freezing artifact by Charles Scouten (Myneurolab website) , titled Tips and Techniques: Freezing Biological Samples to prevent this from happening again. A supporting publication, cited by Scouten, is Jongebloed, W.L., Stokroos, D., Kalicharan, D., and Van der Want, J.J.L. Is Cryopreservation Superior Over Tannic Acid/Arginine/Osmium Tetroxide non-Coating Preparation in Field Emission Scanning Electron Microscopy. Scanning Microscopy 13: 93-109, 1999. This paper might help clarify what damage occurs for SEM even though you might be doing TEM (?). I will be happy to send the article in a separate email if you want it. It would be interesting to have follow up on your results, so keep us posted. Good luck Gayle Callis HTL/HT/MT(ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Re: Accidental freezing of fish samples
Gayle, I would be interested in the articles as well. I had a similar experience: nerve samples from rats were being shipped to me in K2 (which I had previously sent to them). I specifically said not to freeze the samples; they shipped them on dry ice! I was just doing one micron sectioning, but they were useless. Thanks! Peggy -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of gayle callis Sent: Friday, May 20, 2011 12:27 PM To: 'Histonet' Subject: [Histonet] Re: Accidental freezing of fish samples You wrote: Probably the bone staining will be OK - how about sending the student to the arctic circle for a few days in winter? - that may be a good lesson about not freezing tissues. Just kidding :-) On Fri, May 20, 2011 at 9:24 AM, Tora Bardal tora.bardal http://lists.utsouthwestern.edu/mailman/listinfo/histonet @t bio.ntnu.nowrote: One of our students accidentally put his samples in the freezer after formaldehyde/glutaraldehyde/PBS fixation. The samples (fish very early stage, of course rare species) were meant for LM/EM morphology studies. Good morphology is out, but is there a chance he can do cartilage/bone staining? Or do we need to send him abroad for new sampling at the next spawning time? Tora Bardal Senior Engineer NTNU Center of Fisheries and Aquaculture I was amused by Louise's reply. Your comment good morphology is out is correct, especially for EM where bone and cartilage may NOT be optimal after a freeze/ thaw. EM requires optimal fixation and handling to have the best results. There will probably be too much freezing artifact damage (large water ice crystal formation) in the EM samples. However, if doing light microscopy and not worried about soft tissues and with paraffin processing , the bone and cartilage may be ok, but not entirely be ideal ..individual bone and cartilage cells, along with soft tissues, may still show the effects of freezing artifact. These effects may be seen more in cartilage with its higher water content, but give the LM samples a try. You may salvage part of the study. Then send the student out for samples and insist he/she reads up on and understand the damage freezing/thawing can have on fixed samples. I suggest, in lieu of sending your student to the Arctic, that he/she reads the excellent discussion of freezing artifact by Charles Scouten (Myneurolab website) , titled Tips and Techniques: Freezing Biological Samples to prevent this from happening again. A supporting publication, cited by Scouten, is Jongebloed, W.L., Stokroos, D., Kalicharan, D., and Van der Want, J.J.L. Is Cryopreservation Superior Over Tannic Acid/Arginine/Osmium Tetroxide non-Coating Preparation in Field Emission Scanning Electron Microscopy. Scanning Microscopy 13: 93-109, 1999. This paper might help clarify what damage occurs for SEM even though you might be doing TEM (?). I will be happy to send the article in a separate email if you want it. It would be interesting to have follow up on your results, so keep us posted. Good luck Gayle Callis HTL/HT/MT(ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Las Vegas HT Position
Great opportunity for a Histotechnician in a brand new laboratory! We are a 5 physician gastroenterology practice located in Las Vegas, NV looking for a certified HT or HTL. Candidate must meet CLIA grossing requirements. The candidate will be responsible for routine histology duties. This is a full-time position that offers a competitive salary and flexible hours. Interested applicants should contact Meredith Hale, phone (214)596-2219 or e-mail mh...@carisls.com Meredith Hale HT (ASCP) CM Operations Liaison Director and Education Coordinator Caris Life Sciences 6655 North MacArthur Blvd, Irving Texas 75039 direct: 214-596-2219 cell: 469-648-8253 fax: 214-596-7095 mh...@carisls.com mailto:mh...@carisls.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Las Vegas HT Position
Great opportunity for a Histotechnician in a brand new laboratory! We are a 5 physician gastroenterology practice located in Las Vegas, NV looking for a certified HT or HTL. Candidate must meet CLIA grossing requirements. The candidate will be responsible for routine histology duties. This is a part time position that offers a competitive salary and flexible hours. Interested applicants should contact Meredith Hale, phone (214)596-2219 or e-mail mh...@carisls.com Meredith Hale HT (ASCP) CM Operations Liaison Director and Education Coordinator Caris Life Sciences 6655 North MacArthur Blvd, Irving Texas 75039 direct: 214-596-2219 cell: 469-648-8253 fax: 214-596-7095 mh...@carisls.com mailto:mh...@carisls.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC pos. neg. control question
Curt, Thank you. I'm here all week, try the veal. Sincerely, Jay A. Lundgren, M.S., HTL (ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC pos. neg. control question
If an inspector saw that, you would be on immediate suspension. This was the last statement from the original post, I chose not to include it initially but it's just too juicy to keep from everyone else. Having forwarded several of the responses I think he has come to understand that it's not necessarily a requirement that will have us on immediate suspension but rather a preference that, being a private lab, I will gladly provide to keep the business. Thanks again everyone and have a good weekend. Curt From: Jay Lundgren [mailto:jaylundg...@gmail.com] Sent: Friday, May 20, 2011 11:57 AM To: Curt Tague Cc: Ingles Claire; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] IHC pos. neg. control question Curt, Thank you. I'm here all week, try the veal. Sincerely, Jay A. Lundgren, M.S., HTL (ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] SMM-HC
We are using Biocare Smooth Muscle Myosin-Heavy Chains (AP Protocol using Warp Red and previously Vulcan Fast Red)on the Nemesis auto stainer.. Lately we have notice considerable background staining. Any suggestions. The slides almost look like in the old days when you put too much albumin ( as an adhesive) on a slide and did an HE and the eosin coated the slide. We do not notice this with other antibodies. Any suggestions? Diana ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] BrdU on rat tissue
Hi, BrdU from Sigma cat # B2531 works really well. My primary application is in mice, however the target for this antibody is the BrdU that was incorporated into the DNA strand not the animal itself so you should be able to use it in ANY animal. (Don't forget to denature). Good Luck, Amos Message: 2 Date: Thu, 19 May 2011 12:08:38 -0500 From: Michele Wich mw...@7thwavelabs.com Subject: [Histonet] BrdU on rat tissue To: histonet@lists.utsouthwestern.edu Message-ID: 62A8156F8071C8439080D626DF8C33A6017A9D04@wave-mail.7thwave.local Content-Type: text/plain; charset=us-ascii Can anyone recommend a good commercially available kit for BrdU on rat tissue? Thanks in advance for any suggestions. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC pos. neg. control question
Hi, This is simply a question of definitions. They are actually both right. If you have the patient tissue as a negative control you are not using the primary antibody on it but are replacing it (ideally) with an Ig with the same isotype AND dilution (universal negative is stupid). So if your primary is an IgG1 from a mouse and you use it at 1:100, your negative control would be a non-immune mouse serum IgG1 diluted to 1:100. Running this on unrelated material will not show you anything in this case. what it will show you is that the detection system you are using is specific to the antibody you put on the tissue and not to A) something else in the tissue or B) something on the Ig that is not the epitope itself. Although, If you run the same primary antibody on tissue that you expect will not react with the primary antibody, this would prove that the antibody reaction is specific to the antigenin question. This does raise the question of what to do about ubiquitous antigens (like ubiquitin) that are actually everywhere. Every cell has a cell cycle so they will all at one point or another show proliferation or degeneration for example. Does this mean you don't run a negative control? No you need to look at it in the perspective of expected results. There are various ways of using negative (and positive) controls. It would actually be cost prohibitive to run ALL the possible positive and negative controls for every test that we as histologists do. We need to discuss with our pathologists what they want to have done to give them the confidence in our testing to support their diagnosis. But remember that just because another lab and another pathologist does it differently doesn't mean it is wrong. It's just different. Happy Friday Folks, Amos Message: 5 Date: Thu, 19 May 2011 13:25:49 -0400 From: Jean-Martin Lapointe jm.lapoi...@accellab.com Subject: [Histonet] IHC pos. neg. control question To: histonet@lists.utsouthwestern.edu Message-ID: befd613bd39142499989f836556ddc8301272...@ace.accellab.lan Content-Type: text/plain; charset=iso-8859-1 Hi Curt, I agree with your pathologist. The section that you use as a negative control (without primary) in an IHC run should ideally be tissue that is a known positive, and of the same nature as the test sample. Therefore the best sample is often a section of your positive control tissue. The purpose of this negative control is to make sure that any positive staining observed in the test sample is not due to a spurious cross-reaction, unrelated to the primary. I don't think the issue per se is that you are using tissue from the same patient; rather, it is that you are using a sample for which the staining characteristics are not known. For instance, if are using a lymph node section as a negative, for a stain that targets an epithelial marker (eg Her2) in the test breast sample, then your negative tissue is not appropriate, because since lymph node contains no epithelial tissue, it will not stain no matter what. Therefore if your test sample shows a positive reaction in the epithelial tissue, but for some reason that reaction is a spurious false-positive, then the lymph node negative will not reveal that. I realize that this is all very theoretical and hypothetical, but I understand the pathologist wanting to be confident in the knowledge that all potential technical issues are eliminated before making his diagnosis. Jean-Martin ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC pos. neg. control question
Amos Isotype negative controls are based upon protein concentration not dilution. They must be the same protein concentration of the primary antibody at the dilution you are using. Using the same dilution will not work unless both stock solutions (primary antibody and isotype control) are at the same protein concentration which is rarely the case. 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 Amos Brooks Sent: Friday, May 20, 2011 2:16 PM To: jm.lapoi...@accellab.com; histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. neg. control question Hi, This is simply a question of definitions. They are actually both right. If you have the patient tissue as a negative control you are not using the primary antibody on it but are replacing it (ideally) with an Ig with the same isotype AND dilution (universal negative is stupid). So if your primary is an IgG1 from a mouse and you use it at 1:100, your negative control would be a non-immune mouse serum IgG1 diluted to 1:100. Running this on unrelated material will not show you anything in this case. what it will show you is that the detection system you are using is specific to the antibody you put on the tissue and not to A) something else in the tissue or B) something on the Ig that is not the epitope itself. Although, If you run the same primary antibody on tissue that you expect will not react with the primary antibody, this would prove that the antibody reaction is specific to the antigenin question. This does raise the question of what to do about ubiquitous antigens (like ubiquitin) that are actually everywhere. Every cell has a cell cycle so they will all at one point or another show proliferation or degeneration for example. Does this mean you don't run a negative control? No you need to look at it in the perspective of expected results. There are various ways of using negative (and positive) controls. It would actually be cost prohibitive to run ALL the possible positive and negative controls for every test that we as histologists do. We need to discuss with our pathologists what they want to have done to give them the confidence in our testing to support their diagnosis. But remember that just because another lab and another pathologist does it differently doesn't mean it is wrong. It's just different. Happy Friday Folks, Amos Message: 5 Date: Thu, 19 May 2011 13:25:49 -0400 From: Jean-Martin Lapointe jm.lapoi...@accellab.com Subject: [Histonet] IHC pos. neg. control question To: histonet@lists.utsouthwestern.edu Message-ID: befd613bd39142499989f836556ddc8301272...@ace.accellab.lan Content-Type: text/plain; charset=iso-8859-1 Hi Curt, I agree with your pathologist. The section that you use as a negative control (without primary) in an IHC run should ideally be tissue that is a known positive, and of the same nature as the test sample. Therefore the best sample is often a section of your positive control tissue. The purpose of this negative control is to make sure that any positive staining observed in the test sample is not due to a spurious cross-reaction, unrelated to the primary. I don't think the issue per se is that you are using tissue from the same patient; rather, it is that you are using a sample for which the staining characteristics are not known. For instance, if are using a lymph node section as a negative, for a stain that targets an epithelial marker (eg Her2) in the test breast sample, then your negative tissue is not appropriate, because since lymph node contains no epithelial tissue, it will not stain no matter what. Therefore if your test sample shows a positive reaction in the epithelial tissue, but for some reason that reaction is a spurious false-positive, then the lymph node negative will not reveal that. I realize that this is all very theoretical and hypothetical, but I understand the pathologist wanting to be confident in the knowledge that all potential technical issues are eliminated before making his diagnosis. Jean-Martin ___ 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] FW: How to remove DAB to restain with DAB
Hi, There is not any practical way to remove the DAB. I think I read about boiling it in a salt (of some sort) solution removing the DAB. Honestly I never bothered trying it as you need to ask yourself What is this doing to my target antigen?. You should consider just restaining it with the DAB in place using either Alkaline Phosphatase and Fast Red or using a fluorescent detection if the DAB is interfering with the signal. Amos Message: 2 Date: Thu, 19 May 2011 11:55:14 -0500 From: Margaryan, Naira nmargar...@childrensmemorial.org Subject: [Histonet] FW: How to remove DAB to restain with DAB To: histonet-requ...@lists.utsouthwestern.edu histonet-requ...@lists.utsouthwestern.edu Cc: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: c1ba93040c6b9a4a8d84878f93fec36a09b60c5...@cmhexcc01mbx.childrensmemorial.org Content-Type: text/plain; charset=us-ascii Hello, I would like to repeat my DAB staining on the same slide with DAB I run before. I know that acid alcohol will remove hematoxylin but how to remove DAB? I appreciate to any suggestion. Thanks in advance, Naira ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Alizarin Red Troubleshooting
Hi, I seldom do this, but I'll bet I know EXACTLY what the problem is. Try it again and stop the rehydration at 95% ETOH then put it directly into the Alizarin Red (double check the pH of course). This will surely fix it. The calcium gets rinsed out when it is rehydrated. The same thing happens with Von Kossa. I also try to abbreviate rinsing after when dehydrating to coverslipping. Let us know if you still have trouble, Amos Message: 18 Date: Thu, 19 May 2011 09:30:27 -0400 From: Phipps, Amanda amanda.phi...@nationwidechildrens.org Subject: [Histonet] Alizarin Red Troubleshooting To: 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu Message-ID: 20CE7246011C12489BA04BEB85CCDA1F0351BF252B@res2k7ms01 Content-Type: text/plain; charset=us-ascii Hi All We are currently trying to stain for calcium deposits using Alizarin Red. A few months ago our protocol worked fine, but this time we are getting zero staining. Reagent has been made fresh, and pH is between 4.1 and 4.2. Here is our protocol. Deparaffinize to distilled H20 Stain slides with Alizarin Red S for 30 seconds to 5 minutes and observe reaction microscopically Shake off excess dye and blot sections Dehydrate in acetone 10 seconds to 20 seconds, then acetone-xylene (1:1) solution 10-20 seconds Clear in xylene and mount in synthetic mounting medium. Any help is greatly appreciated!! Amanda Phipps, HTL (ASCP) Histotechnologist Morphology Core Lab The Research Institute at Nationwide Children's Hospital 700 Children's Drive Columbus, Ohio 43205 (614) 355-3474 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC pos. neg. control question
Thanks Liz, You are absolutly right there, but have you ever noticed some folks eyes glaze over when you say that they must both be run at the same ug/mL? I would be lying if I said it never happened to me until I forced myself to work it out. Once you do though it isn't too bad. Unfortunately people don't think about this much. They want to put a slide on a machine and walk away without thinking about what they are doing. I guess I was oversimplifying the description. Thanks for pointing that out as it is actually an important aspect to the dilutions. Amos On Fri, May 20, 2011 at 4:25 PM, Liz Chlipala l...@premierlab.com wrote: Amos Isotype negative controls are based upon protein concentration not dilution. They must be the same protein concentration of the primary antibody at the dilution you are using. Using the same dilution will not work unless both stock solutions (primary antibody and isotype control) are at the same protein concentration which is rarely the case. 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 Amos Brooks Sent: Friday, May 20, 2011 2:16 PM To: jm.lapoi...@accellab.com; histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. neg. control question Hi, This is simply a question of definitions. They are actually both right. If you have the patient tissue as a negative control you are not using the primary antibody on it but are replacing it (ideally) with an Ig with the same isotype AND dilution (universal negative is stupid). So if your primary is an IgG1 from a mouse and you use it at 1:100, your negative control would be a non-immune mouse serum IgG1 diluted to 1:100. Running this on unrelated material will not show you anything in this case. what it will show you is that the detection system you are using is specific to the antibody you put on the tissue and not to A) something else in the tissue or B) something on the Ig that is not the epitope itself. Although, If you run the same primary antibody on tissue that you expect will not react with the primary antibody, this would prove that the antibody reaction is specific to the antigenin question. This does raise the question of what to do about ubiquitous antigens (like ubiquitin) that are actually everywhere. Every cell has a cell cycle so they will all at one point or another show proliferation or degeneration for example. Does this mean you don't run a negative control? No you need to look at it in the perspective of expected results. There are various ways of using negative (and positive) controls. It would actually be cost prohibitive to run ALL the possible positive and negative controls for every test that we as histologists do. We need to discuss with our pathologists what they want to have done to give them the confidence in our testing to support their diagnosis. But remember that just because another lab and another pathologist does it differently doesn't mean it is wrong. It's just different. Happy Friday Folks, Amos Message: 5 Date: Thu, 19 May 2011 13:25:49 -0400 From: Jean-Martin Lapointe jm.lapoi...@accellab.com Subject: [Histonet] IHC pos. neg. control question To: histonet@lists.utsouthwestern.edu Message-ID: befd613bd39142499989f836556ddc8301272...@ace.accellab.lan Content-Type: text/plain; charset=iso-8859-1 Hi Curt, I agree with your pathologist. The section that you use as a negative control (without primary) in an IHC run should ideally be tissue that is a known positive, and of the same nature as the test sample. Therefore the best sample is often a section of your positive control tissue. The purpose of this negative control is to make sure that any positive staining observed in the test sample is not due to a spurious cross-reaction, unrelated to the primary. I don't think the issue per se is that you are using tissue from the same patient; rather, it is that you are using a sample for which the staining characteristics are not known. For instance, if are using a lymph node section as a negative, for a stain that targets an epithelial marker (eg Her2) in the test breast sample, then your negative tissue is not appropriate, because since lymph node contains no epithelial tissue, it will not stain no matter what. Therefore if your test sample shows a positive reaction in the epithelial tissue, but for some reason that reaction is a spurious false-positive, then the lymph node negative will not reveal that. I realize that this is all very theoretical and hypothetical, but I understand the pathologist wanting to be confident
RE: [Histonet] IHC pos. neg. control question
No problem Amos, I have a one page handout that I use for workshops and presentations on how to do the math on this, it can be tricky, so if anyone is interested just e-mail me. Since we are talking about matched isotype negative controls, essentially three things need to be taken into consideration: 1. The isotype of the primary antibody 2. The protein concentration of the working dilution of the primary antibody 3. The antibody form - (affinity purified, culture supernatant, ascities fluid, etc.) the isotype negative control needs to match the primary antibody - some spec sheets list appropriate isotype negative control reagents some do not There is one caveat that I would like to address for those that are working with animal tissues - you need to check the spec sheet on your negative control reagents to make sure that it does not cross react with the species you are working with. If it does then you may get staining in your negative control slide. Here is a table that was generated from one of the older versions of the dako handbook that addresses the antibody form and suggested negative control reagents. Primary Antibody Type Suggested Negative Control Reagent Monoclonal Mouse - produced in ascites Antibody produced in ascites, same isotype as the primary antibody OR Normal nonimmune mouse serum Monoclonal Mouse - produced in tissue culture Antibody produced in tissue culture, same isotype as the primary antibody Polyclonal Rabbit or Goat - immunoglobulin fraction Normal rabbit or goat immunoglobulin fraction Solid-phase absorbed Polyclonal Rabbit - immunoglobulin fraction Rabbit immunoglobulin fraction - solid phase absorbed Polyclonal Rabbit - whole serum Normal or nonimmune rabbit serum, whole serum Hope this helps, and everyone have a great weekend 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: Amos Brooks [mailto:amosbro...@gmail.com] Sent: Friday, May 20, 2011 3:05 PM To: Liz Chlipala Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] IHC pos. neg. control question Thanks Liz, You are absolutly right there, but have you ever noticed some folks eyes glaze over when you say that they must both be run at the same ug/mL? I would be lying if I said it never happened to me until I forced myself to work it out. Once you do though it isn't too bad. Unfortunately people don't think about this much. They want to put a slide on a machine and walk away without thinking about what they are doing. I guess I was oversimplifying the description. Thanks for pointing that out as it is actually an important aspect to the dilutions. Amos On Fri, May 20, 2011 at 4:25 PM, Liz Chlipala l...@premierlab.com wrote: Amos Isotype negative controls are based upon protein concentration not dilution. They must be the same protein concentration of the primary antibody at the dilution you are using. Using the same dilution will not work unless both stock solutions (primary antibody and isotype control) are at the same protein concentration which is rarely the case. Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC PO Box 18592 Boulder, Colorado 80308 office (303) 682-3949 tel:%28303%29%20682-3949 fax (303) 682-9060 tel:%28303%29%20682-9060 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 Amos Brooks Sent: Friday, May 20, 2011 2:16 PM To: jm.lapoi...@accellab.com; histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC pos. neg. control question Hi, This is simply a question of definitions. They are actually both right. If you have the patient tissue as a negative control you are not using the primary antibody on it but are replacing it (ideally) with an Ig with the same isotype AND dilution (universal negative is stupid). So if your primary is an IgG1 from a mouse and you use it at 1:100, your negative control would be a non-immune mouse serum IgG1 diluted to 1:100. Running this on unrelated material will not show you anything in this case. what it will show you is that the detection system you are using is specific to the antibody you put on the tissue and not to A) something else in the tissue or B) something on the Ig that is not the epitope itself. Although, If you run the same primary antibody on tissue that you expect will not react with the primary antibody, this would prove that the antibody reaction is specific to the antigenin question. This does raise the question of what to do about ubiquitous antigens (like ubiquitin) that are actually everywhere. Every cell has a
