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