Re: [Histonet] blocking endogenous activities
Peroxidase, the enzyme you want to block with hydrogen peroxide, is a protein and, as such, should be cross-linked by formalin if the tissue is properly sixed. In consequence, you should eliminate the crosslinkage to fully expose the enzyme before blocking it. Therefore, you should do HIER first and block the peroxidase afterwards. On the other hand, many labs do René J. --- On Thu, 3/1/12, Tuyen Nguyen wrote: From: Tuyen Nguyen Subject: [Histonet] blocking endogenous activities To: histonet@lists.utsouthwestern.edu Date: Thursday, March 1, 2012, 6:48 PM In my lab, in IHC protocols for most antibodies, hydrogen peroxide is applied before antigen heat retrieval. However, I wonder there is any different result in IHC stain if hydrogen peroxide for blocking endogenous activities is applied after antigen heat retrieval by using steamer or pressure cooker? Thank you, Mai Nguyen Truong Research Associate II Los Angeles, California ___ 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] blocking endogenous activities
You need to do it sometime before the HRP-labeled conjugate is put on. If you put it on after the HRP conjugate, you'll block the HRP substrate and won't see where the primary antibody is binding. I usually do mine before antigen retrieval, but have done it after the primary antibody step on frozen sections and cell smears. Jan Shivers Senior Scientist IHC/Histology/EM Veterinary Diagnostic Lab University of Minnesota St. Paul, MN On Thu, Mar 1, 2012 at 5:55 PM, Bea DeBrosse-Serra < bdebrosse-se...@isisph.com> wrote: > The majority of the time we do it after retrieval. I believe you can do > the hydrogen peroxide block at any point, as long as you do it before the > DAB. > > Beatrice DeBrosse-Serra HT(ASCP)QIHC > Isis Pharmaceuticals > Antisense Drug Discovery > 2588 Gazelle Ct. > Carlsbad, CA 92010 > 760-603-2371 > > > > -Original Message- > From: histonet-boun...@lists.utsouthwestern.edu [mailto: > histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tuyen Nguyen > Sent: Thursday, March 01, 2012 3:49 PM > To: histonet@lists.utsouthwestern.edu > Subject: [Histonet] blocking endogenous activities > > In my lab, in IHC protocols for most antibodies, hydrogen peroxide is > applied before antigen heat retrieval. However, I wonder there is any > different result in IHC stain if hydrogen peroxide for blocking endogenous > activities is applied after antigen heat retrieval by using steamer or > pressure cooker? > Thank you, > Mai Nguyen Truong > Research Associate II > Los Angeles, California > ___ > 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
RE: [Histonet] blocking endogenous activities
The majority of the time we do it after retrieval. I believe you can do the hydrogen peroxide block at any point, as long as you do it before the DAB. Beatrice DeBrosse-Serra HT(ASCP)QIHC Isis Pharmaceuticals Antisense Drug Discovery 2588 Gazelle Ct. Carlsbad, CA 92010 760-603-2371 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tuyen Nguyen Sent: Thursday, March 01, 2012 3:49 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] blocking endogenous activities In my lab, in IHC protocols for most antibodies, hydrogen peroxide is applied before antigen heat retrieval. However, I wonder there is any different result in IHC stain if hydrogen peroxide for blocking endogenous activities is applied after antigen heat retrieval by using steamer or pressure cooker? Thank you, Mai Nguyen Truong Research Associate II Los Angeles, California ___ 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] blocking endogenous activities
In my lab, in IHC protocols for most antibodies, hydrogen peroxide is applied before antigen heat retrieval. However, I wonder there is any different result in IHC stain if hydrogen peroxide for blocking endogenous activities is applied after antigen heat retrieval by using steamer or pressure cooker? Thank you, Mai Nguyen Truong Research Associate II Los Angeles, California ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
