RE: [Histonet] RE: Eliminating the edge effect in IHC/IF
Actually, when I read about the problem, my thought was that if you don't circle your tissue with an immunopen or wax pencil, then the reagents (esp. primary antibody)might not cover the tissue evenly as you say. So I tend to agree with you. Peggy -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of C.M. van der Loos Sent: Tuesday, January 12, 2010 2:55 PM To: histonet@lists.utsouthwestern.edu Cc: k...@prosci-inc.com Subject: [Histonet] RE: Eliminating the edge effect in IHC/IF Hi all,We also have observed this phenomenon many times. But sorry Colleen and Rene, I don't believe that an fixation issue is the explanation why the edges are sometimes stronger than the rest. To my opinion this is a bit too easy. One of my explanations is that the immuno reagents tend to stick to the edges of the tissue section, especially when no Tween20 is involved. As a result the outer edges might become a little dry during incubation times and cause darker mostly non-specific staining. Always try to cover a section completely, including a rim around the section. However, to be honest, I am sure my explanation is certainly not always appropriate. Anyone elseCheers,ChrisChris van der Loos, PhD Dept. of Pathology Academic Medical Center M2-230 Meibergdreef 9 NL-1105 AZ Amsterdam The Netherlands phone: +31 20 5665631 From: Rene J Buesa rjbu...@yahoo.com Subject: Re: [Histonet] Eliminating the edge effect in IHC/IF To: histonet@lists.utsouthwestern.edu, Karen Cai k...@prosci-inc.com Usually that is the result of incomplete fixation. Check your fixation protocol. Ren J. --- On Mon, 1/11/10, Karen Cai k...@prosci-inc.com wrote: From: Karen Cai k...@prosci-inc.com Subject: [Histonet] Eliminating the edge effect in IHC/IF To: histonet@lists.utsouthwestern.edu Date: Monday, January 11, 2010, 2:00 PM Hi, I have a question for the generous input. When I do the IHC or IF, it seems very common that the intensity of the edge area of the tissue is always stronger than the central tissue part. Is it possible to eliminate this and make the staining evenly distributed around the whole tissue section? Your kind help is greatly appreciated, Thanks in advance, Best, Karen Karen Cai Research Scientist Prosci Incorporated (858) 513-2638 x 204 (858) 513-2692 Fax http://www.prosci-inc.com www.prosci-inc.com ___ 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
RE: [Histonet] RE: Eliminating the edge effect in IHC/IF
Again, provided that you are doing IHC manually. René J. --- On Tue, 1/12/10, Sherwood, Margaret msherw...@partners.org wrote: From: Sherwood, Margaret msherw...@partners.org Subject: RE: [Histonet] RE: Eliminating the edge effect in IHC/IF To: C.M. van der Loos c.m.vanderl...@amc.uva.nl, histonet@lists.utsouthwestern.edu Cc: k...@prosci-inc.com Date: Tuesday, January 12, 2010, 3:28 PM Actually, when I read about the problem, my thought was that if you don't circle your tissue with an immunopen or wax pencil, then the reagents (esp. primary antibody)might not cover the tissue evenly as you say. So I tend to agree with you. Peggy -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of C.M. van der Loos Sent: Tuesday, January 12, 2010 2:55 PM To: histonet@lists.utsouthwestern.edu Cc: k...@prosci-inc.com Subject: [Histonet] RE: Eliminating the edge effect in IHC/IF Hi all,We also have observed this phenomenon many times. But sorry Colleen and Rene, I don't believe that an fixation issue is the explanation why the edges are sometimes stronger than the rest. To my opinion this is a bit too easy. One of my explanations is that the immuno reagents tend to stick to the edges of the tissue section, especially when no Tween20 is involved. As a result the outer edges might become a little dry during incubation times and cause darker mostly non-specific staining. Always try to cover a section completely, including a rim around the section. However, to be honest, I am sure my explanation is certainly not always appropriate. Anyone elseCheers,ChrisChris van der Loos, PhD Dept. of Pathology Academic Medical Center M2-230 Meibergdreef 9 NL-1105 AZ Amsterdam The Netherlands phone: +31 20 5665631 From: Rene J Buesa rjbu...@yahoo.com Subject: Re: [Histonet] Eliminating the edge effect in IHC/IF To: histonet@lists.utsouthwestern.edu, Karen Cai k...@prosci-inc.com Usually that is the result of incomplete fixation. Check your fixation protocol. Ren J. --- On Mon, 1/11/10, Karen Cai k...@prosci-inc.com wrote: From: Karen Cai k...@prosci-inc.com Subject: [Histonet] Eliminating the edge effect in IHC/IF To: histonet@lists.utsouthwestern.edu Date: Monday, January 11, 2010, 2:00 PM Hi, I have a question for the generous input. When I do the IHC or IF, it seems very common that the intensity of the edge area of the tissue is always stronger than the central tissue part. Is it possible to eliminate this and make the staining evenly distributed around the whole tissue section? Your kind help is greatly appreciated, Thanks in advance, Best, Karen Karen Cai Research Scientist Prosci Incorporated (858) 513-2638 x 204 (858) 513-2692 Fax http://www.prosci-inc.com www.prosci-inc.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] RE: Eliminating the edge effect in IHC/IF
I think we'll agree that there are different scenarios so that the solution is not a one size fits all. For example, darkened staining around the periphery of needle core biopsies is not uncommon with even tinctorial stains, often thought to be the result of drying of the tissue during the collection of the sample. Years ago I came across an article maintaining that the dark staining on the periphery of needle cores was in fact due to the trauma of the needle cutting into the sampled organ. I've since forgotten the author's name and wish I could get my hands on that reference. So I agree with Chris that there doesn't appear to be one simple answer to prevent this artifact and while fixation may contribute in some circumstances it's unlikely to be the remedy for all. Vinnie Della Speranza Manager for Anatomic Pathology Services 165 Ashley Avenue Suite 309 Charleston, SC 29425 tel. 843-792-6353 fax. 843-792-8974 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of C.M. van der Loos Sent: Tuesday, January 12, 2010 2:55 PM To: histonet@lists.utsouthwestern.edu Cc: k...@prosci-inc.com Subject: [Histonet] RE: Eliminating the edge effect in IHC/IF Hi all,We also have observed this phenomenon many times. But sorry Colleen and Rene, I don't believe that an fixation issue is the explanation why the edges are sometimes stronger than the rest. To my opinion this is a bit too easy. One of my explanations is that the immuno reagents tend to stick to the edges of the tissue section, especially when no Tween20 is involved. As a result the outer edges might become a little dry during incubation times and cause darker mostly non-specific staining. Always try to cover a section completely, including a rim around the section. However, to be honest, I am sure my explanation is certainly not always appropriate. Anyone elseCheers,ChrisChris van der Loos, PhD Dept. of Pathology Academic Medical Center M2-230 Meibergdreef 9 NL-1105 AZ Amsterdam The Netherlands phone: +31 20 5665631 From: Rene J Buesa rjbu...@yahoo.com Subject: Re: [Histonet] Eliminating the edge effect in IHC/IF To: histonet@lists.utsouthwestern.edu, Karen Cai k...@prosci-inc.com Usually that is the result of incomplete fixation. Check your fixation protocol. Ren� J. --- On Mon, 1/11/10, Karen Cai k...@prosci-inc.com wrote: From: Karen Cai k...@prosci-inc.com Subject: [Histonet] Eliminating the edge effect in IHC/IF To: histonet@lists.utsouthwestern.edu Date: Monday, January 11, 2010, 2:00 PM Hi, I have a question for the generous input. When I do the IHC or IF, it seems very common that the intensity of the edge area of the tissue is always stronger than the central tissue part. Is it possible to eliminate this and make the staining evenly distributed around the whole tissue section? Your kind help is greatly appreciated, Thanks in advance, Best, Karen Karen Cai Research Scientist Prosci Incorporated (858) 513-2638 x 204 (858) 513-2692 Fax http://www.prosci-inc.com www.prosci-inc.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] RE: Eliminating the edge effect in IHC/IF
I do my immuno staining manually using ProbeOn Plus slides which employ the capillary action principle. I always monitor the taking up/whicking off of my solutions for each pair of slides throughout the entire staining process and know that my sections never dry out (I incubate using moist chambers with plenty of fluid). I work at a research/diagnostic medical school facility where our primary focus is on experimental tissues, using standardized and rigid guidelines for fixation: 24 hours at 4 degrees overnight in 4% PF, followed by post fixation in 70% ethanol for a few days. Never see the edge effect with these sections. However, I do occasionally see the effect using archival diagnostic samples that were fixed in 10% NBF for a minimum of 48 hours, and probably longer. I have therefore always perceived this effect to be caused by extended, rather than incomplete fixation. Hermina -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa Sent: Tuesday, January 12, 2010 3:41 PM To: C.M. van der Loos; histonet@lists.utsouthwestern.edu; MargaretSherwood Cc: k...@prosci-inc.com Subject: RE: [Histonet] RE: Eliminating the edge effect in IHC/IF Again, provided that you are doing IHC manually. René J. --- On Tue, 1/12/10, Sherwood, Margaret msherw...@partners.org wrote: From: Sherwood, Margaret msherw...@partners.org Subject: RE: [Histonet] RE: Eliminating the edge effect in IHC/IF To: C.M. van der Loos c.m.vanderl...@amc.uva.nl, histonet@lists.utsouthwestern.edu Cc: k...@prosci-inc.com Date: Tuesday, January 12, 2010, 3:28 PM Actually, when I read about the problem, my thought was that if you don't circle your tissue with an immunopen or wax pencil, then the reagents (esp. primary antibody)might not cover the tissue evenly as you say. So I tend to agree with you. Peggy -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of C.M. van der Loos Sent: Tuesday, January 12, 2010 2:55 PM To: histonet@lists.utsouthwestern.edu Cc: k...@prosci-inc.com Subject: [Histonet] RE: Eliminating the edge effect in IHC/IF Hi all,We also have observed this phenomenon many times. But sorry Colleen and Rene, I don't believe that an fixation issue is the explanation why the edges are sometimes stronger than the rest. To my opinion this is a bit too easy. One of my explanations is that the immuno reagents tend to stick to the edges of the tissue section, especially when no Tween20 is involved. As a result the outer edges might become a little dry during incubation times and cause darker mostly non-specific staining. Always try to cover a section completely, including a rim around the section. However, to be honest, I am sure my explanation is certainly not always appropriate. Anyone elseCheers,ChrisChris van der Loos, PhD Dept. of Pathology Academic Medical Center M2-230 Meibergdreef 9 NL-1105 AZ Amsterdam The Netherlands phone: +31 20 5665631 From: Rene J Buesa rjbu...@yahoo.com Subject: Re: [Histonet] Eliminating the edge effect in IHC/IF To: histonet@lists.utsouthwestern.edu, Karen Cai k...@prosci-inc.com Usually that is the result of incomplete fixation. Check your fixation protocol. Ren J. --- On Mon, 1/11/10, Karen Cai k...@prosci-inc.com wrote: From: Karen Cai k...@prosci-inc.com Subject: [Histonet] Eliminating the edge effect in IHC/IF To: histonet@lists.utsouthwestern.edu Date: Monday, January 11, 2010, 2:00 PM Hi, I have a question for the generous input. When I do the IHC or IF, it seems very common that the intensity of the edge area of the tissue is always stronger than the central tissue part. Is it possible to eliminate this and make the staining evenly distributed around the whole tissue section? Your kind help is greatly appreciated, Thanks in advance, Best, Karen Karen Cai Research Scientist Prosci Incorporated (858) 513-2638 x 204 (858) 513-2692 Fax http://www.prosci-inc.com www.prosci-inc.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
