Re: [Histonet] IHC DAB
Hi Kristy, As the others said, so many things that could be on the way...But making sure your system is based on a HRP enzyme is definitely a good start, as mentioned by Joe. If you can share details, I might be able to help too. Welcome to the IHC world! Cheers, Ana Ana Maluenda Research Assistant/Laboratory Manager Atherothrombosis and Vascular Biology Laboratory From: Joe Myers Sent: Saturday, 2 May 2020 4:49 AM To: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] IHC DAB Kristy: I’m thinking along the same lines as Paula; is it possible that your detection reagent contains only Alkaline Phosphatase (ALP) as there a reactive enzyme? If so, a peroxide/DAB solution simply won’t react with it. Can’t wait to see your protocol, with detailed descriptions of the antigen, pretreatment reagent and ‘labeling’ enzyme. Cheers, Joe Myers, M.S.,CT/QIHC(ASCP) From: Kristy Castillo via Histonet Sent: Friday, May 1, 2020 11:00 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC DAB Hi Histonetters, We are starting our IHC (fun times), we are having trouble with the DAB lighting up. Processed for 5 thru 10 minutes and still nothing. Our permanent red is working just fine. Any ideas. Thank you! Kristy Protecting your privacy is important to us. The Baker Heart and Diabetes Institute will handle your information in accordance with the Privacy Act 1988 (Cth) and its Privacy Policy which is available at www.baker.edu.au or on request by contacting priv...@baker.edu.au or by calling 1800 838 498. The Privacy Policy also explains how you can access and correct your personal information, or make a complaint about a breach of the Australian Privacy Principles. bidipp2014.0.1a -- Message protected by MailGuard: e-mail anti-virus, anti-spam and content filtering.http://www.mailguard.com.au/mg ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC DAB
Kristy: I’m thinking along the same lines as Paula; is it possible that your detection reagent contains only Alkaline Phosphatase (ALP) as there a reactive enzyme? If so, a peroxide/DAB solution simply won’t react with it. Can’t wait to see your protocol, with detailed descriptions of the antigen, pretreatment reagent and ‘labeling’ enzyme. Cheers, Joe Myers, M.S.,CT/QIHC(ASCP) From: Kristy Castillo via Histonet Sent: Friday, May 1, 2020 11:00 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC DAB Hi Histonetters, We are starting our IHC (fun times), we are having trouble with the DAB lighting up. Processed for 5 thru 10 minutes and still nothing. Our permanent red is working just fine. Any ideas. Thank you! Kristy ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC DAB
Since you are newbies, I am just putting IHC 101 out there. Permanent red is for alkaline phosphatase detection and DAB is for HRP. Paula Keene Pierce, BS, HTL(ASCP)HTPresidentExcalibur Pathology, Inc.5830 N Blue Lake DriveNorman, OK 73069PH 405-759-3953http://www.excaliburpathology.com A sharp knife is nothing without a sharp eye. - Klingon Proverb On Friday, May 1, 2020, 10:29:00 AM CDT, Kristy Castillo via Histonet wrote: Hi Histonetters, We are starting our IHC (fun times), we are having trouble with the DAB lighting up. Processed for 5 thru 10 minutes and still nothing. Our permanent red is working just fine. Any ideas. Thank you! Kristy ___ 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] IHC DAB
Thanks for the tip! On Fri, May 1, 2020 at 8:53 AM Val Tolley wrote: > It’s difficult to know without your full protocol, but you can easily test > your DAB. > > Take a paper towel and add a drop of your secondary/HRP. Add a drop of > DAB to that. If it doesn’t immediately turn brown, your hydrogen peroxide > has likely degraded into water. Make a fresh batch of DAB and try again. > > > > > On May 1, 2020, at 8:28 AM, Kristy Castillo via Histonet < > histonet@lists.utsouthwestern.edu> wrote: > > > > Hi Histonetters, > > > > We are starting our IHC (fun times), we are having trouble with the DAB > > lighting up. Processed for 5 thru 10 minutes and still nothing. Our > > permanent red is working just fine. Any ideas. > > > > Thank you! > > > > Kristy > > ___ > > 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] IHC DAB
It’s difficult to know without your full protocol, but you can easily test your DAB. Take a paper towel and add a drop of your secondary/HRP. Add a drop of DAB to that. If it doesn’t immediately turn brown, your hydrogen peroxide has likely degraded into water. Make a fresh batch of DAB and try again. > On May 1, 2020, at 8:28 AM, Kristy Castillo via Histonet > wrote: > > Hi Histonetters, > > We are starting our IHC (fun times), we are having trouble with the DAB > lighting up. Processed for 5 thru 10 minutes and still nothing. Our > permanent red is working just fine. Any ideas. > > Thank you! > > Kristy > ___ > 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] IHC DAB
Hydrogen Peroxide gone bad / or not being added??? From: Kristy Castillo via Histonet Sent: Friday, May 1, 2020 11:00 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC DAB Hi Histonetters, We are starting our IHC (fun times), we are having trouble with the DAB lighting up. Processed for 5 thru 10 minutes and still nothing. Our permanent red is working just fine. Any ideas. Thank you! Kristy ___ Histonet mailing list Histonet@lists.utsouthwestern.edu https://urldefense.com/v3/__http://lists.utsouthwestern.edu/mailman/listinfo/histonet__;!!HXCxUKc!nqBcFt8qtbMg7kzneedVpPwolLzEfsp6AKUFOKtTOI2wNvOmwykUTuORZ3qW4g$ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC DAB
Hi Histonetters, We are starting our IHC (fun times), we are having trouble with the DAB lighting up. Processed for 5 thru 10 minutes and still nothing. Our permanent red is working just fine. Any ideas. Thank you! Kristy ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
