Hugh thanks for your reply and I'll type my answers in blue below after the questions:
_____ From: Hugh Luk [mailto:hlu...@msn.com] Sent: Thursday, February 03, 2011 6:21 PM To: plu...@biopath.org Subject: RE: [Histonet] TTF-1/background staining Paula, Incomplete IHC staining has many factors. 1) What type of antigen retrieval are you using? We use the Lab Vision's Citrate Buffer Ph 6 Can you use a high pH solution instead? Unmasking hard epitopes (TTF-1 can be stubborn) will look better under a higher pH AR (or TR for Dako users). Are you re-using your solution? Please say "No" "NO" or at least "not often." Lastly how are your retrieving? We use Biocare's Decloaking Chamber, basically a pressure cooker Pressure HIER or rolling boil methods work best for TTF1. 2) What type of buffer are you using. We use TBS with Tween added: a small capful to a 5 liter container TBS or PBS should both work. Is it pH'd correctly? Since you automate, does your buffer contain Tween 20? Too much or too little could ruin your day. 3) Primary antibody. Concentrate? It's a predilute Could your diluent (solution) be faulty or your titre incorrect? Call or email Cell Marque to help you with this? I did call Cell Marque and they gave me some suggestions such as using an EDTA based retrieval solution and to reduce the antibody time to 15 minutes, and to also increase the blocker from 5 to 10 minutes Another email from Tony asks about dilution too, and hints at problems with the anitbody (AR). I use Dako's version of TTF-1 for human tissues, and I have not seen a problem. Perhaps you could call Dako and procure a free sample? I'll try that 4) Fixation. TTF-1 isn't known to be fixation-dependent, but how long are your tissues sitting in formalin before processing? It depends.we receive samples from the hospital, and so it can vary. The smaller samples are in formalin at least 6 hours: includes the sample sitting in the formalin container waiting for gross, plus then the processor time Problems with over or under fixation could cause your description. Also, what fixative are you using? We use 10% Formalin I have heard various fixatives being used on liver biopsies: Carnoy's, Helly's, Hollende's, Zinc formalin, ect. If you are using another fixative, can you go back to 10% neutral buffered formalin? 5) Tissue processor. How is your tissue processor? It's a VIP and the paraffin is checked at 60 degrees Could your paraffin be overheating? 6) Detection system. I have simply never tried your kit, but I will say several antibodies from Thermo have worked well. Thermo lists their polymer system as non-avidin-biotin based, and should be ultra sensitive. If it works on everything else (especially stains that visualize the nucleus), I would say your kit is not the problem. But it still might not like your antibody. For example, the nuclear epitope is rather small and Dako's polymer kit (Envision plus) was a large bio-synthesized molecule that many people thought would sterically hinder a good percentage of binding, reducing the signal. Don't know if that's true, it works okay for us. 7) 30 minute primary incubation should be fine. Is there any chance your slides are drying while incubating? I'm not 100% sure..the slides look to me like they are not drying out. We apply the buffer as soon as we place the slides on the machine, and then we keep the cover to the machine closed while it's running, so I'm assuming the slides aren't drying out We sometimes put paper towels on the bottom of the stainer to assure the chamber is not drying out our slides (but that's only when we get REALLY paranoid). 8) Liver. Is dirty. Debbie has a point that the liver may be working against you. You could try a protein block, or something that Thermo may suggest to deter background. Do you ink your liver biopsies? No we don't ink the liver bx's Davidson's inks used to give us some problems with IHC on small tissues (so we went to marking with eosin or hematoxylin). 9) Liver does not normally stain TTF-1. HCC's should be negative. Lung would stain well (unless your "Lung" tissue is also a metastisis), but for example breast mets could be focal and weak to diffuse and strong. It depends on the tumor. Could the tumor be of this type? I will ask the pathologist this question Good luck and feel free to email me if something I wrote needs to be clarified. I apologize for the rambling on... don't apologize! I really appreciate this Hugh Cancer Research center of Hawaii > From: dsi...@statlab.com > To: plu...@biopath.org; histonet@lists.utsouthwestern.edu > Date: Thu, 3 Feb 2011 16:02:22 -0600 > Subject: RE: [Histonet] TTF-1/background staining > CC: > > Hi Paula, > > I am probably reading between the lines here but on your TTF protocol do you use a streptavidin biotin detection system and if so do you block for endogenous biotin, liver will have endogenous biotin where lung may not have as much? thanks > > Debbie Siena HT(ASCP)QIHC > Technical Manager | StatLab Medical Products > Direct: 972-436-1010 x229 > > > -----Original Message----- > From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Paula Lucas > Sent: Thursday, February 03, 2011 2:38 PM > To: histonet@lists.utsouthwestern.edu > Subject: [Histonet] TTF-1/background staining > > How can I eliminate the background or as you say, non-specific staining? > > > > I'm no expert here, I admit, and so I'm asking for your help and > suggestions. I have searched the Histonet archives, and a lot of what I'm > seeing deals with animal or rodent tissues, and a lot of it was confusing to > me. > > > > To give you a little background info: > > We use the Lab Vision stainer and the TTF-1 antibody we use is from Cell > Marque. We were having issues with this marker from Lab Vision, so we > switched to TTF-1 a while ago. We use the UltraVision LP detection kit from > Lab Vision. It's a polymer driven detection kit. > > > > The antibody is a ready to use, and we have the time set at 30 minutes. > > > > We were getting the background staining on a liver specimen last week, and > we also had this problem today on a lung case. My doctor is getting > frustrated, and wants me to do something about this, and to repeat the TTF-1 > stain on the lung, so if someone can give me some suggestions to try, I'm > ready to try them. > > > > Thanks in advance, > > Paula > > Lab Manager > > Bio-Path Medical Group > > Fountain Valley, CA > > _______________________________________________ > 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