Re: [Histonet] [ SOLVED ][ Histonet ] Strange circles in IHC slides
Hello, I'm glad that you already solved the problem your way. I didn't read your first post, but we had exactly the same problem (and we're also using cover plates). This artifact is very likely caused by tiny air bubbles which are trapped under the cover plate. The crucial step is when you drip a little buffer onto the plate in order to get your slide in proper position. You get much lesser air bubbles if a) no detergent is used and b) the PBS or TBS is at room temperature and not cold (which isn't good anyway). We don't use detergents anymore (on coverplates). Alexandra Meinl Dr. Alexandra Meinl Ludwig Boltzmann Institute for Experimental and Clinical Traumatology Austrian Cluster for Tissue Regeneration Histology Donaueschingenstrasse 13 1200 Vienna - Austria Contact @ Bernhard Gottlieb University School of Dentistry, Waehringerstr. 25a, A-1090 Vienna tel: +43 1 4277 67026 fax: +43 1 4277 67019 email: alexandra.me...@trauma.lbg.ac.at ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] [ SOLVED ][ Histonet ] Strange circles in IHC slides
Are you sure that you don't introduce air bubbles when you put your slides into the coverplates? The antibody will not touch the tissue if there is an air bubble. Willem Hoekert Van: histonet-boun...@lists.utsouthwestern.edu namens Rene J Buesa Verzonden: do 25-2-2010 16:42 Aan: histonet@lists.utsouthwestern.edu; histonet.nos...@vneubert.com Onderwerp: Re: [Histonet] [ SOLVED ][ Histonet ] Strange circles in IHC slides To me it seems that the sections after being picked from the water bath were not completely drained and the dewaxing process was incomplete in a way that the "round" areas kept certain amount of paraffin wax that prevented the reagents reactions. The fact that the areas are round are an indication that water was involved, since water always leave a round imprint, due to its surface tension. I would suggest that you dewax the sections with a 2% aq. solution of dish washer soap. Dewaxing with xylene sections containing water will be incomplete because it does not mix completely with water but the detergent will mix with the water and will better remove the paraffin.René J. --- On Thu, 2/25/10, histonet.nos...@vneubert.com wrote: From: histonet.nos...@vneubert.com Subject:>[Histonet] [ SOLVED ][ Histonet ] Strange circles in IHC slides To: histonet@lists.utsouthwestern.edu Date: Thursday, February 25, 2010, 8:34 AM Hello Histonet, it has been a while (~10 months) since I posted a problem about uneven immuno-staining with specimen showing unstained circles after manual staining with HRP-polymere/DAB method; complete mail see below, response mails see Histonet archive (via website). Links to pictures I took: http://img12.imageshack.us/img12/8513/ts0402162049.jpg http://img13.imageshack.us/img13/6514/ts0402162104.jpg The problem occured suddenly, without having changed any reagents or methods. Things I changed to avoid the unstained spots: *Adding 0,05% Tween 20 to TBS *Blocking peroxidase in coplin jar, not mounted in racks *Lowering antibody concentration which temporarily produced better results. After reviewing a big number of slides it showed up that most of the tissue affected was lung, liver and kidney which mostly means a lot of blood in the tissue when fixation in formalin starts. Erythrocytes, granulocytes and macrophages show a lot of endogenous and pseudoendogenous peroxidase activity. This is how it's done since then: Slides are taken from racks into a big coplin jar with 3% H2O2 diluted in distilled water (demineralized H2O). A slow magnetic stirrer on the bottom of the jar keeps the solution floating around the tissue, removing any O2 bubble that might appear. Slides then are remounted and rinsed a lot with TBS-T. Thank you for all your help, though it's a little late... V. Neubert, Germany - Original Message - From: histonet.nos...@vneubert.com To: histonet@lists.utsouthwestern.edu Date: 02.04.2009 18:12:18 Subject: [Histonet] Strange circles in IHC slides [...]> http://img12.imageshack.us/img12/8513/ts0402162049.jpg > http://img13.imageshack.us/img13/6514/ts0402162104.jpg [...]> > So, has ever anyone experienced sth. like this? > My conjugate control (every step except the antibody) was fine, nothing > to be seen about DAB and no circles at all. > > I used Shandon single-use coverplates, sterile buffer, fresh antibody > aliquots. Any idea? > > Thanks, > > V. Neubert ___ 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 Disclaimer: Dit e-mail bericht is uitsluitend bestemd voor de geadresseerde(n). Verstrekking aan en gebruik door anderen dan geadresseerden is niet toegestaan. Indien u niet de geadresseerde bent, wordt u verzocht de verzender hiervan op de hoogte te stellen en het bericht te verwijderen. In verband met electronische verzending kunnen aan dit e-mail bericht geen rechten worden ontleend. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] [ SOLVED ][ Histonet ] Strange circles in IHC slides
To me it seems that the sections after being picked from the water bath were not completely drained and the dewaxing process was incomplete in a way that the "round" areas kept certain amount of paraffin wax that prevented the reagents reactions. The fact that the areas are round are an indication that water was involved, since water always leave a round imprint, due to its surface tension. I would suggest that you dewax the sections with a 2% aq. solution of dish washer soap. Dewaxing with xylene sections containing water will be incomplete because it does not mix completely with water but the detergent will mix with the water and will better remove the paraffin.René J. --- On Thu, 2/25/10, histonet.nos...@vneubert.com wrote: From: histonet.nos...@vneubert.com Subject:>[Histonet] [ SOLVED ][ Histonet ] Strange circles in IHC slides To: histonet@lists.utsouthwestern.edu Date: Thursday, February 25, 2010, 8:34 AM Hello Histonet, it has been a while (~10 months) since I posted a problem about uneven immuno-staining with specimen showing unstained circles after manual staining with HRP-polymere/DAB method; complete mail see below, response mails see Histonet archive (via website). Links to pictures I took: http://img12.imageshack.us/img12/8513/ts0402162049.jpg http://img13.imageshack.us/img13/6514/ts0402162104.jpg The problem occured suddenly, without having changed any reagents or methods. Things I changed to avoid the unstained spots: *Adding 0,05% Tween 20 to TBS *Blocking peroxidase in coplin jar, not mounted in racks *Lowering antibody concentration which temporarily produced better results. After reviewing a big number of slides it showed up that most of the tissue affected was lung, liver and kidney which mostly means a lot of blood in the tissue when fixation in formalin starts. Erythrocytes, granulocytes and macrophages show a lot of endogenous and pseudoendogenous peroxidase activity. This is how it's done since then: Slides are taken from racks into a big coplin jar with 3% H2O2 diluted in distilled water (demineralized H2O). A slow magnetic stirrer on the bottom of the jar keeps the solution floating around the tissue, removing any O2 bubble that might appear. Slides then are remounted and rinsed a lot with TBS-T. Thank you for all your help, though it's a little late... V. Neubert, Germany - Original Message - From: histonet.nos...@vneubert.com To: histonet@lists.utsouthwestern.edu Date: 02.04.2009 18:12:18 Subject: [Histonet] Strange circles in IHC slides [...]> http://img12.imageshack.us/img12/8513/ts0402162049.jpg > http://img13.imageshack.us/img13/6514/ts0402162104.jpg [...]> > So, has ever anyone experienced sth. like this? > My conjugate control (every step except the antibody) was fine, nothing > to be seen about DAB and no circles at all. > > I used Shandon single-use coverplates, sterile buffer, fresh antibody > aliquots. Any idea? > > Thanks, > > V. Neubert ___ 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
