[Histonet] TGF beta on FFPE
Hello Histonet, I'd like to know if anyone of you has experience with TGF beta receptor and TGF beta ligand IHC on FFPE tissue. Any input will be highly appreciated :) Thanks a lot V. Neubert ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: [ Histonet ] Tris Buffer Preservative
I used to sterilze my TBS by autoclaving it. For aliquots I used TBS with 0,1% sodium acide. I never had any problems with bacteria; my 10x TBS concentrate was fine for weeks. - Original Message - From: amosbro...@gmail.com To: histonet@lists.utsouthwestern.edu Date: 24.02.2010 17:59:45 Subject: [Histonet] Tris Buffer Preservative Hi, I have been making up my own tris buffer for immunohistochemistry. It has been working swimmingly, with one exception. I have been doing some work on Salmonella, and apparently the researcher has been seeing bacteria in her immunofluorescent slides that she says are moving and seem to be viable. She wanted to culture all my reagents. So the primary and secondary antibody diluents (commercially purchased) came up clean, but the TBS ended up with a pretty healthy growing colony. Now since I don't do IHC overnight in an incubator, I don't think this is necessarily a catastrophy. (No one else has noticed this) It does seem to warrant further investigation though. So for the folks that make their own solutions up, what do you use as a preservative for your buffers and how much do you use. I haven't seen anything in any of the recipes I have found. I was thinking Sodium Azide, but it is really hazardous, and Wikipedia says it is actually explosive ( http://en.wikipedia.org/wiki/Sodium_azide). Biocare Medical's data sheet says they use less than 0.25% procyclin. (thanks for not hiding the ingredients Biocare, I love you for that!) Has anyone tried that? Any other suggestions would be welcome. Thanks, Amos ___ 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] [ SOLVED ][ Histonet ] Strange circles in IHC slides
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] Re: [ Histonet ] Hematoxylin
Gill II (ThermoFisher) 5 min, 3x1 min A. Dest. That's it :) I need some pointers on how to intensify the hematoxylin on the H and E stain. Is there a way to do that? I currently use Richard Allan hematoxylin 7211. The H is weak. All reagents were changed out last night. Any of your expertise would be of great help. Thank you guys in advance. Delia ___ 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
